EA044359B1 - METHODS AND APPLICATION OF SYNTHETIC OLIGOSACCHARIDE PREPARATION TO INCREASE THE EFFICIENCY OF ANIMAL NUTRITION - Google Patents

Description

Related applications

This application claims priority to U.S. Provisional Patent Application No.

62/757438, filed November 8, 2018, and US Provisional Patent Application No. 62/757439, filed November 8, 2018, the disclosures of each of which are incorporated herein by reference in their entirety.

Prior Art

Global population growth places constant pressure on agriculture and livestock production to increase yields and sustainability of livestock products. Meeting this demand requires continued improvement in the performance of animals raised for protein. For example, increasing nutritional efficiency allows an animal to achieve the same weight or performance with less feed intake than a comparable animal with lower nutritional efficiency. Because feed production requires the consumption of resources and energy to obtain, prepare, and transport its ingredients, improving animal growth performance reduces the amount of resources and energy required to raise an animal. Additionally, since feed is the largest cost of raising animals, improving feed efficiency provides economic benefits to the producer.

Feed additives have been developed to improve live weight gain and nutritional efficiency of animals. For example, antibiotic growth promoters (AGPs) have become widely used due to their ability to increase weight gain and feed efficiency. However, growing pressure from regulators and consumers has forced the livestock industry to move away from antibiotic feed additives that exhibit a poor resistance profile. In particular, non-therapeutic use of antibiotics contributes to increased microbial resistance and multidrug resistance in strains of dangerous pathogens.

Microbially derived feed products (DFMs) have been explored in livestock production as an alternative to AGPs. Unlike antibiotics, DFMs aim to support the animal host by providing types of probiotics that have a positive effect on the animal's digestive system. DFMs, such as commensal bacteria or yeasts, are typically limited to spore-forming microbes so that they can withstand dry product preparation and incorporation into conventional feed manufacturing processes. However, it is difficult to prepare probiotic organisms as dry products without compromising the viability of the active strains. The resulting dose variability, that is, the number of viable organisms delivered to the digestive tract, leads to instability in commercial production conditions. In addition, many of the most prominent commensal gastrointestinal microorganisms, that is, the taxa with the greatest positive impact on growth performance, are either non-spore forming or non-culturable, making it virtually impossible to convert them into a stable feed additive.

Thus, there is a need for new feed additives and nutritional compositions that improve animal growth, nutritional efficiency and health, including feed additives that promote the growth of beneficial gastrointestinal microorganisms that are difficult to incorporate into DFM feed additives or inhibit the growth of harmful gastrointestinal microorganisms. intestinal microorganisms.

Summary of the invention

The present invention is directed to a method of increasing the body weight of an animal, including administering to the animal a nutritional composition containing a basic nutritional composition and a synthetic oligosaccharide preparation containing at least n fractions of oligosaccharides, each of which has a different degree of polymerization selected from 1 to n (fractions from DP1 to DPn), where n is an integer greater than 3 and where each of the fractions DP1 and DP2 independently comprises from 0.5 to 15% oligosaccharides containing anhydrosubunits, as measured by mass spectrometry.

According to the invention, the body weight of the animal is at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 g greater than the body weight of the animal before the application of the nutritional composition containing the synthetic oligosaccharide preparation, when measured after at least 30 days, 35 days, 40 days, 45 days, 50 days, 60 days, 70 days, 80 days, or 90 days after the first administration of a nutritional composition containing a synthetic oligosaccharide preparation, and wherein the animal takes the nutritional composition at least once during every 24 hours

The present invention is also directed to a method for reducing the feed conversion ratio of an animal, comprising administering to the animal a nutritional composition comprising a basic nutritional composition and a synthetic oligosaccharide preparation containing at least n oligosaccharide fractions, each of which has a different degree of polymerization selected from 1 to n (fractions DP1 to DPn), where n is an integer greater than 3-containing and where each of the fractions DP1 and DP2 independently comprises from 0.5 to 15% oligosaccharides containing anhydro subunits, as measured by relative abundance by mass spectrometry.

- 1 044359

According to the invention, the feed conversion ratio (FCR) of the animal is reduced by at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10% compared to the FCR of the animal before the use of a nutritional composition containing a synthetic oligosaccharide preparation, when measured at least 30 days, 35 days, 40 days, 45 days, 50 days, 60 days, 70 days, 80 days, or 90 days after the first administration of a nutritional composition containing a synthetic oligosaccharide preparation, and wherein the animal receives the nutritional composition at at least once every twenty-four hours.

The present invention is also directed to a method for increasing the nutritional efficiency of an animal, comprising administering to the animal a nutritional composition containing a basic nutritional composition and a synthetic oligosaccharide preparation containing at least n fractions of oligosaccharides, each of which has a different degree of polymerization selected from 1 to n (fractions from DP1 to DPn), where n is an integer greater than 3 containing and where each of the fractions DP1 and DP2 independently comprises from 0.5 to 15% oligosaccharides containing anhydrosubunits, as measured by relative abundance by mass spectrometry, where nutritional efficiency is the ratio animal product to the amount of feed consumed.

According to the invention, the animal's nutritional efficiency is increased by at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10% compared to the animal's nutritional efficiency before the use of a nutritional composition containing a synthetic oligosaccharide preparation.

According to the invention, the animal's nutritional efficiency is increased by at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10% compared to the animal's nutritional efficiency before the use of a nutritional composition containing a synthetic oligosaccharide preparation, when measured by at least at least 30 days, 35 days, 40 days, 45 days, 50 days, 60 days, 70 days, 80 days, or 90 days after the first administration of a nutritional composition containing a synthetic oligosaccharide preparation, and where the animal ingests the nutritional composition at least one once every 24 hours.

According to the invention, the nutritional composition contains at least 100 ppm, 200 ppm, 300 ppm, 400 ppm, 500 ppm, 600 ppm, 700 ppm, 800 ppm, 900 ppm, 1000 ppm, 1500 ppm or 2000 ppm oligosaccharide preparation.

The present invention is also directed to a method of modulating the growth of at least one species of microorganisms in the gastrointestinal tract of an animal, comprising administering to the animal a nutritional composition containing a basic nutritional composition and a synthetic oligosaccharide preparation containing at least n fractions of oligosaccharides, each of which has a different degree polymerization selected from 1 to n (fractions DP1 to DPn), where n is an integer greater than 3-containing and where each of fractions DP1 and DP2 independently comprises from 0.5 to 15% oligosaccharides containing anhydrosubunits, by relative abundance, measured by mass spectrometry.

According to the invention, at least one type of microorganism is an archaea, bacteria, protozoa, virus, bacteriophage, parasite or fungus.

According to the invention, the method includes stimulating the growth of at least one type of microorganism, and wherein the level of at least one type of microorganism in the gastrointestinal sample is increased compared to the level of at least one type of microorganism in the gastrointestinal sample from a comparable control animal in which a comparable a nutritional composition that does not contain a synthetic oligosaccharide drug.

The present invention is also directed to a method of modulating the expression of at least one microbial protein in the gastrointestinal tract of an animal, comprising administering to the animal a nutritional composition comprising a basic nutritional composition and a synthetic oligosaccharide preparation containing at least n oligosaccharide fractions, each of which has a different degree polymerization selected from 1 to n (fractions DP1 to DPn), where n is an integer greater than 3 containing and where each of fractions DP1 and DP2 independently comprises from 0.5 to 15% oligosaccharides containing anhydrosubunits, by relative abundance, measured by mass - spectrometry.

According to the invention, the level of at least one microbial protein in the gastrointestinal sample is reduced compared to the level of at least one microbial protein in the gastrointestinal sample from the animal prior to application of the nutritional composition to the animal.

The present invention is also directed to the use of a nutritional composition comprising a basic nutritional composition and a synthetic oligosaccharide preparation containing at least n oligosaccharide fractions, each of which has a different degree of polymerization selected from 1 to n (fractions DP1 to DPn), where n is an integer greater than 3-containing and wherein each of fractions DP1 and DP2 independently comprises from 0.5 to 15% oligosaccharides containing anhydrosubunits, as measured by mass spectrometry, to increase the body weight of the animal.

The present invention is also directed to the use of a nutritional composition comprising a basic nutritional composition and a synthetic oligosaccharide preparation containing at least n oligosaccharide fractions, each of which has a different degree of polymerization selected from 1 to n (fractions DP1 to DPn) , where n is an integer greater than 3 containing and where each of the DP1 and DP2 fractions independently comprises from 0.5 to 15% oligosaccharides containing anhydrosubunits, by relative abundance, as measured by mass spectrometry, reducing the animal's feed conversion ratio.

The present invention is also directed to the use of a nutritional composition comprising a basic nutritional composition and a synthetic oligosaccharide preparation containing at least n oligosaccharide fractions, each of which has a different degree of polymerization selected from 1 to n (fractions DP1 to DPn), where n is an integer greater than 3-containing and wherein each of the DP1 and DP2 fractions independently comprises from 0.5 to 15% anhydrosubunit-containing oligosaccharides, as measured by mass spectrometry, to improve the nutritional efficiency of the animal.

The present invention is also directed to the use of a nutritional composition comprising a basic nutritional composition and a synthetic oligosaccharide preparation containing at least n oligosaccharide fractions, each of which has a different degree of polymerization selected from 1 to n (fractions DP1 to DPn), where n is an integer greater than 3-containing and wherein each of fractions DP1 and DP2 independently comprises from 0.5 to 15% oligosaccharides containing anhydrosubunits, as measured by relative abundance by mass spectrometry, to modulate the growth of at least one species of microorganisms in the gastrointestinal animal tract.

The present invention is also directed to the use of a nutritional composition comprising a basic nutritional composition and a synthetic oligosaccharide preparation containing at least n oligosaccharide fractions, each of which has a different degree of polymerization selected from 1 to n (fractions DP1 to DPn), where n is an integer greater than 3-containing and wherein each of fractions DP1 and DP2 independently comprises from 0.5 to 15% oligosaccharides containing anhydrosubunits, as measured by relative abundance by mass spectrometry, to modulate the expression of at least one microbial protein in the gastrointestinal animal tract.

Brief description of the figures

The new features of the invention are set out in detail in the accompanying claims. A better understanding of the features and advantages of the present invention will be obtained with reference to the following detailed description, which sets forth illustrative embodiments employing the principles of the invention and the accompanying drawings (also figures and figs herein), wherein: FIG. 1 illustrates part of the 1H, 13C-HSQC NMR spectrum of oligosaccharide drug 9.2;

fig. 2 illustrates the MALDI-MS spectrum of the oligosaccharide preparation from Example 9.7, which demonstrates the presence of anhydrosubunits;

fig. 3 illustrates the 1D ^-proton NMR spectrum of the anhydro-DP1 component from the oligosaccharide preparation of Example 9;

fig. 4 illustrates the 1D APT ¹³ C-NMR spectrum of the anhydro-DP1 component from the oligosaccharide preparation of Example 9;

fig. 5 shows an enlargement of the GC-MS chromatogram (TIC and XIC plots (m/z 229)) for the oligosaccharide preparation of Example 2.9 after derivatization;

fig. 6 illustrates the 1H,13C-HSQC spectrum of an oligosaccharide drug with an anhydrous caramelization subunit;

fig. 7 illustrates a portion of MALDI-MS spectra comparing the oligosaccharide formulation of Example 9 at various laser energies;

fig. 8A shows LC-MS/MS detection of anhydro-DP2 forms at concentrations of 1-80 μg/ml oligosaccharide drug in water; fig. 8B illustrates the linear calibration curve obtained from the LC-MS/MS detection of FIG. 8A;

fig. 9 shows quantification of anhydro-DP2 content in various control and treated nutritional compositions;

fig. 10 illustrates a 2D-1H JRES NMR spectrum of a sample of gluco-oligosaccharides containing anhydrosubunits;

fig. 11 is a typical 1H,13C-HSQC NMR spectrum of a sample of gluco-oligosaccharides containing anhydrosubunits, with corresponding resonances and assignments used for bond assignment;

fig. 12 illustrates an overlay of the 1H DOSY spectra of three oligosaccharides containing anhydrosubunits.

fig. 13 illustrates a comparison of 1,6-anhydro-β-D-glucose (DP1-18), 1,6-anhydro-β-O-cellobiose (DP2-18) and a sample of oligosaccharides containing anhydrosubunits;

fig. 14 illustrates mass chromatograms of oligosaccharides containing anhydrosubunits (top) and digested oligosaccharides containing anhydrosubunits (bottom) under selective multiple reaction monitoring (MRM);

fig. 15 shows a graph of relative content versus degree of polymerization

- 3 044359 (DP) of the oligosaccharide from example 9. The graph shows that the oligosaccharide preparation has a monotonically decreasing DP distribution;

fig. 16 shows a plot of relative abundance versus degree of polymerization of the oligosaccharide from Example 9. The plot shows that the oligosaccharide preparation has a non-monotonically decreasing DP distribution;

fig. 17 shows the species (taxa) of microorganisms in the cecal microbiota associated with increased growth rates measured by body weight;

fig. 18 shows the species (taxa) of microorganisms in the cecal microbiota associated with reduced growth rates measured by body weight;

fig. 19 is a graph showing the increase in ex vivo transcription of hydrolytic digestive enzymes by microbiota produced with the oligosaccharide preparation of Example 9;

fig. 20 is a graph showing the effect of the oligosaccharide preparation of Example 9 on the functional metagenomics of piglets;

fig. 21 shows a decrease in the number of undesirable representatives of the phylum Proteobacteria in the intestinal flora of commercial broiler chickens in the experimental group (birds fed treated diets containing the oligosaccharide drug) compared to the control group (birds fed control diets without the oligosaccharide drug);

fig. 22 is a graph showing the correlation between the relative abundance of Escherichia coli (left) and other Escherichia species (right) and mortality of commercial broiler chickens;

fig. 23 shows two oligosaccharides containing anhydrosubunits from the DP1 fraction and one from the DP2 fraction;

fig. 24 illustrates an oligosaccharide containing anhydrosubunits (cellotriosan);

fig. 25A shows the MALDI-MS spectrum of the oligosaccharide preparation from Example 2, which demonstrates the presence of anhydrosubunits; fig. 25B illustrates the MALDI-MS spectrum of the oligosaccharide preparation from Example 2, which demonstrates the presence of anhydrosubunits;

fig. 26A illustrates the detection by LC-MS/MS of the anhydro-DP2, anhydroDP1 and DP2 species of the oligosaccharide drug from Example 1; fig. 26B illustrates the detection by LC-MS/MS of the anhydro-DP2, anhydro-DP1 and DP2 species of the oligosaccharide drug from Example 1; fig. 26C illustrates the detection by LC-MS/MS of the anhydro-DP2, anhydroDP1 and DP2 species of the oligosaccharide drug from Example 1;

fig. 27A illustrates the detection by LC-MS/MS of the anhydro-DP2, anhydroDP1 and DP2 species of the oligosaccharide drug from Example 3; fig. 27B illustrates the detection by LC-MS/MS of the anhydro-DP2, anhydro-DP1 and DP2 species of the oligosaccharide drug from Example 3; fig. 27C illustrates the detection by LC-MS/MS of the anhydro-DP2, anhydroDP1 and DP2 species of the oligosaccharide drug from Example 3;

fig. 28A illustrates the detection by LC-MS/MS of the anhydro-DP2, anhydroDP1 and DP2 species of the oligosaccharide drug from Example 4; fig. 28B illustrates the detection by LC-MS/MS of the anhydro-DP2, anhydro-DP1 and DP2 species of the oligosaccharide drug from Example 4; fig. 28C illustrates the detection by LC-MS/MS of the anhydro-DP2, anhydroDP1 and DP2 species of the oligosaccharide drug from Example 4;

fig. 29A illustrates the detection by LC-MS/MS of the anhydro-DP2, anhydroDP1 and DP2 species of the oligosaccharide drug from Example 7; fig. 29B illustrates the detection by LC-MS/MS of the anhydro-DP2, anhydro-DP1 and DP2 species of the oligosaccharide drug from Example 7; fig. 29C illustrates the detection by LC-MS/MS of the anhydro-DP2, anhydroDP1 and DP2 species of the oligosaccharide drug from Example 7;

fig. 30A illustrates the spectrum from GC-MS detection with the anhydro-DP2, anhydro-DP1 and DP2 fractions of the oligosaccharide drug from Example 1; fig. 30B illustrates the increase in DP2 and anhydro DP2 fractions as shown in FIG. 30A;

fig. 31A illustrates the spectrum from GC-MS detection with the anhydro-DP2, anhydro-DP1 and DP2 fractions of the oligosaccharide drug from Example 3; fig. 31B illustrates the increase in DP2 and anhydro DP2 fractions as shown in FIG. 31A;

fig. 32A illustrates the spectrum from GC-MS detection with the anhydro-DP2, anhydro-DP1 and DP2 fractions of the oligosaccharide drug from Example 4; fig. 32B illustrates the increase in DP2 and anhydro DP2 fractions as shown in FIG. 32A;

fig. 33A illustrates the spectrum from GC-MS detection with the anhydro-DP2, anhydro-DP1 and DP2 fractions of the oligosaccharide drug from Example 7; fig. 33B illustrates the increase in DP2 and anhydro DP2 fractions as shown in FIG. 33A;

fig. 34 illustrates the effect of reaction temperature, water content and reaction time on the amount of oligosaccharides containing DP2 anhydrosubunits in oligosaccharide preparations compared to the oligosaccharide preparation according to Example 2;

- 4 044359 fig. 35 illustrates the NMR assignments of 1,6-anhydro-beta-D-glucofuranose and 1,6-anhydro-beta-D-glucopyranose;

fig. 36 illustrates MALDI-MS spectra comparing the oligosaccharide formulation of Example 9 at different laser energies.

Detailed Description of the Invention

The following description and examples illustrate in detail embodiments of the present invention. It should be understood that this present invention is not limited to the specific embodiments described in this application, and as such may vary. Those skilled in the art will appreciate that there are numerous variations and modifications of this present invention that fall within its scope.

All terms are intended to be understood as they would be understood by one skilled in the art. Unless otherwise specified, all technical and scientific terms used in this application have the same meaning as commonly understood by one skilled in the art to which the invention relates.

The section headings used in this application are for organizational purposes only and should not be construed as limiting the subject matter described.

Although various features of the present invention may be described in the context of a single embodiment, these features may also be provided separately or in any suitable combination. Conversely, although the present invention may be described herein in the context of individual embodiments for the sake of clarity, the present invention may also be practiced in a single embodiment.

The following definitions are in addition to those in the art and are specific to the current application and are not intended to apply to any related or unrelated case, such as any general patent or application. Although any methods and materials similar or equivalent to those described herein may be practiced for testing the present invention, preferred materials and methods are described herein. Accordingly, the terminology used herein is intended to describe specific embodiments only and is not intended to be limiting.

I. Definitions

The terminology used in this application is intended to describe specific cases only and is not intended to be limiting. The singular forms used herein are intended to include the plural forms unless the context clearly indicates otherwise. Moreover, to the extent that the terms including, includes, having, has, with, or variations thereof are used in the detailed description and/or claims, such terms are intended to be included in the same manner as the term containing.

It is clear that terms such as contains, containing, etc. have the meaning ascribed to them under the United States Patent Laws; those. they mean includes, included, including, etc. and are intended to include or extend, and do not exclude, additional, not listed elements or method steps; and that such terms as consisting essentially of and consisting essentially of have the meaning ascribed to them in the United States Patent Law; that is, they admit elements not explicitly listed, but exclude elements that appear in the prior art or that affect the essential or novel characteristics of the invention.

The term and/or, as used herein in a phrase such as A and/or B, is intended to include both A and B; A or B; A (separately); and B (separately). Likewise, the term and/or, when used in a phrase such as A, B and/or C, is intended to cover each of the following embodiments: A, B and C; A, B or C; A or B; A or C; B or C; A and B; A and C; B and C; A (separately); B (separately) and C (separately).

When ranges of physical properties, such as molecular weight, or chemical properties, such as chemical formulas, are used herein, all combinations and subcombinations of ranges and specific embodiments are intended to be included therein. The term approximately when referring to a number or numerical range means that the number or numerical range stated is approximate within experimental variability (or within statistical experimental error), and thus the number or numerical range will in some cases vary from 1 to 15 % of the specified number or number range. In some embodiments, the term approximately means within 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1 %, or 0.05% of the set value or range.

As used herein, the term use includes providing the synthetic oligosaccharide preparation, nutritional composition, liquid or animal feed composition described herein to an animal such that the animal can consume the synthetic oligosaccharide preparation, nutritional composition, liquid or animal feed composition. In such embodiments, the animal ingests a portion of the synthetic oligosaccharide drug, nutritional composition, or animal feed composition. IN

- 5 044359 In some embodiments, the animal is provided with a synthetic oligosaccharide preparation, nutritional composition, liquid or animal feed composition such that the animal can consume the synthetic oligosaccharide preparation, nutritional composition, liquid or animal feed composition as desired. In some embodiments, the synthetic oligosaccharide drug, nutritional composition, liquid, or animal feed composition is administered to said animal as a prescribed diet. In some embodiments, the synthetic oligosaccharide drug, nutritional composition, liquid, or animal feed composition is administered to said animal through manual feeding, such as oral syringe feeding, tube feeding, etc. In some embodiments, the synthetic oligosaccharide drug, nutritional composition, liquid, or animal feed composition is administered orally to the animal, such as by self-administration or hand feeding. In some embodiments, the animal ingests a portion of the synthetic oligosaccharide drug, nutritional composition, liquid, or animal feed composition every 24 hours or every second day for at least 7 days, 14 days, 21 days, 30 days, 45 days, 60 days , 75 days, 90 days or 120 days. In some embodiments, the oligosaccharide drug may be dissolved in water or other liquid, and the animal absorbs some of the oligosaccharide drug by drinking the liquid. In some embodiments, the oligosaccharide is delivered to the animal through drinking water. In some embodiments, the oligosaccharide preparation, nutritional composition, liquid, or animal feed composition is consumed as desired.

As used herein, the term inclusion level or dose refers to the concentration of the oligosaccharide drug in the nutritional composition, liquid, diet or animal feed composition provided to the animal. In some embodiments, the inclusion level is measured as the mass concentration of the oligosaccharide drug in the final nutritional composition, liquid, diet, or animal feed. For example, the inclusion level may be measured in parts per million (ppm) of the oligosaccharide on a dry solids basis per total weight of the final nutritional composition, liquid, diet or animal feed. In some embodiments, the dry solids weight of the oligosaccharide preparation is measured as the weight of the DP1+ species on a dry matter basis. In other embodiments, the dry solids weight of the oligosaccharide preparation is measured as the weight of the DP2+ species on a dry matter basis.

As used herein, the term specific dose refers to the amount of oligosaccharide drug consumed by an animal per unit of time and relative to its body weight. In some embodiments, the specific dose may be measured in units of mg of oligosaccharide preparation (dry matter basis) per kg of animal body weight per day (i.e., mg/kg/day).

As used herein, the term feed conversion ratio (FCR) means the ratio of the mass of feed input (eg, consumed by an animal) to animal product, wherein the animal product is the target animal product. For example, the animal product for dairy animals is milk, while the animal product for meat animals is body weight.

As used herein, the term nutritional efficiency refers to the ratio of an animal product to the amount of feed consumed (eg, consumed by the animal), the animal product being the target animal product.

As used herein, the term anhydrosubunit refers to the thermal dehydration product of a monosaccharide (or monosaccharide subunit) or the caramelization product of a sugar. For example, the anhydrosubunit may be an anhydromonosaccharide such as anhydroglucose. As another example, an anhydrosubunit may be linked to one or more regular or anhydromonosaccharide subunits via a glycosidic bond.

The term oligosaccharide refers to a monosaccharide or compound containing two or more monosaccharide subunits linked by glycosidic bonds. Essentially, an oligosaccharide includes a conventional monosaccharide, an anhydromonosaccharide, or a compound containing two or more monosaccharide subunits, where one or more monosaccharide subunits are optionally independently replaced by one or more anhydrosaccharide subunits. The oligosaccharide may be functionalized. As used herein, the term oligosaccharide covers all types of oligosaccharides wherein each monosaccharide subunit in the oligosaccharide is independently and optionally functionalized and/or replaced by a corresponding anhydromonosaccharide subunit.

As used herein, the term oligosaccharide drug refers to a drug that contains at least one oligosaccharide.

As used herein, the term gluco-oligosaccharide refers to glucose or a compound containing two or more glucose monosaccharide subunits linked by glycosidic bonds. Essentially, a glucooligosaccharide includes glucose, anhydroglucose, or a compound containing two or more glucose monosaccharide subunits linked by glycosidic bonds, each of said glucose monosaccharide subunits being optionally and independently replaced by a subunit

- 6 044359 anhydroglucose.

As used herein, the term galactooligosaccharide refers to galactose or a compound containing two or more galactose monosaccharide subunits linked by glycosidic bonds. Essentially, a galactooligosaccharide includes galactose, anhydrogalactose, or a compound containing two or more galactose monosaccharide subunits linked by glycosidic bonds, wherein each of said galactose monosaccharide subunits is optionally and independently replaced by an anhydrogalactose subunit.

As used herein, the term glucogalacto-oligosaccharide preparation refers to a composition that is obtained by the reaction of complete or partial condensation of the sugars glucose and galactose. Accordingly, in some embodiments, the glucogalactose-oligosaccharide preparation includes glucooligosaccharides, galactooligosaccharides, compounds containing one or more glucose monosaccharide subunits and one or more galactose monosaccharide subunits linked by glycosidic bonds, or a combination thereof. In some embodiments, the gluco-oligosaccharide preparation includes gluco-oligosaccharides and compounds containing one or more glucose monosaccharide subunits and one or more galactose monosaccharide subunits linked by glycosidic bonds. In some embodiments, the glucogalactose-oligosaccharide preparation includes galactooligosaccharides and compounds containing one or more glucose monosaccharide subunits and one or more galactose monosaccharide subunits linked by glycosidic bonds. In some embodiments, the glucogalactose-oligosaccharide preparation includes compounds comprising one or more glucose monosaccharide subunits and one or more galactose monosaccharide subunits linked by glycosidic bonds.

As used herein, the terms monosaccharide unit and monosaccharide subunit are used interchangeably. A monosaccharide subunit refers to the monomer of a monosaccharide in an oligosaccharide. For an oligosaccharide having a degree of polymerization of 1, the oligosaccharide may be referred to as a monosaccharide subunit or monosaccharide. For an oligosaccharide having a degree of polymerization of 2 or higher, its monosaccharide subunits are linked by glycosidic bonds.

As used herein, the term regular monosaccharide refers to a monosaccharide that does not contain an anhydrous subunit. The term regular disaccharide refers to a disaccharide that does not contain an anhydrous subunit. Accordingly, the term regular subunit refers to a subunit that is not an anhydrosubunit.

As used herein, the term anhydro-DPn oligosaccharide, anhydro-DPn species, or DPn anhydrosubunit-containing oligosaccharide refers to an oligosaccharide that has a degree of polymerization n and includes anhydrosubunits. Essentially, anhydroglucose is an oligosaccharide containing the DP1 anhydrosubunit, and cellotriosan is an oligosaccharide containing the DP3 anhydrosubunit.

The term relative abundance or prevalence as used herein refers to the prevalence of a species in terms of how common or rare the species is. For example, the DP1 fraction comprising 10% of oligosaccharides containing anhydro subunits, by relative abundance, may refer to a plurality of DP1 oligosaccharides, where 10% of DP1 oligosaccharides are anhydromonosaccharides. The relative abundance, for example, for a certain fraction of DP oligosaccharides, can be determined using suitable analytical tools, for example, mass spectrometry and liquid chromatography, such as LC-MS/MS, GC-MS, HPLC-MS and MALDI-MS. In some embodiments, relative abundance is determined by integrating the area under the peaks on chromatographs (eg, LC-MS/MS, GC-MS, and HPLC-MS) that correspond to the fractions of interest. In some embodiments, relative abundance is determined from peak intensities (eg, MALDI-MS). In some embodiments, the relative abundance is determined by a combination of analytical methods, such as determination of mass after separation using liquid chromatography.

As used herein, the singular number includes the plural unless the context clearly dictates otherwise. Thus, for example, a reference to the term agent includes a plurality of such agents, and a reference to an oligosaccharide includes a reference to one or more oligosaccharides (or a plurality of oligosaccharides) and equivalents thereof known to those skilled in the art, and so on.

II. Oligosaccharide drug

Disclosed herein are oligosaccharide preparations suitable for use in nutritional compositions. In some embodiments, said oligosaccharide preparation contains at least n oligosaccharide fractions, each having a different degree of polymerization selected from 1 to n (fractions DP1 to DPn), where n is an integer equal to 2 or more. In some embodiments, n is an integer greater than 2. In some embodiments, each of the fractions 1 to n in the oligosaccharide preparation independently comprises from 0.1 to 90% of the oligosaccharides containing anhydrosubunits, as measured by relative abundance using mass spectrometry. In some embodiments, the relative abundance of oligosaccharides in each fraction decreases monotonically with the degree of polymerization.

In some embodiments, n is an integer equal to 3 or more. In some embodiments, n is an integer ranging from 1 to 100, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or 50. In some embodiments, each of fractions 1 to n in the oligosaccharide preparation independently comprises from 0.1 to 90% oligosaccharides, containing anhydro subunits, by relative abundance measured by mass spectrometry or LC-MS/MS or GC-MS. In some embodiments, each of fractions 1 to n in the oligosaccharide preparation independently comprises from about 0.1 to 15% oligosaccharides containing anhydrosubunits. In some embodiments, each of fractions 1 to n in the oligosaccharide preparation independently comprises from about 0.5 to 15% oligosaccharides containing anhydrosubunits. In some embodiments, each fraction from DP1 and DP2 independently comprises from about 0.1 to 15% oligosaccharides containing anhydrosubunits, as measured by relative abundance by mass spectrometry, such as MALDI-MS, or LC-MS/MS or GC-MS. In some embodiments, each fraction from DP1 and DP2 independently comprises from about 0.5 to 15% oligosaccharides containing anhydrosubunits. In some embodiments, each fraction of DP1 and DP2 independently comprises about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.8%, 1%, 2%, or 3% to about 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% anhydrosubunit-containing oligosaccharides, by relative abundance measured by mass spectrometry, LC-MS/MS, or GC-MS . In some embodiments, the relative abundance of oligosaccharides in each fraction decreases monotonically with the degree of polymerization.

In some embodiments, the oligosaccharide drug is a synthetic oligosaccharide drug. In some embodiments, a synthetic oligosaccharide preparation refers to a plurality of oligosaccharides produced by a process that does not require living organisms. In some embodiments, a synthetic oligosaccharide preparation refers to a variety of oligosaccharides produced through a process that does not require enzymes. In some embodiments, a synthetic oligosaccharide drug refers to a variety of chemically produced oligosaccharides. In some embodiments, a synthetic oligosaccharide drug refers to a variety of oligosaccharides obtained by condensation of sugars.

A. Prebiotic value of oligosaccharides

Disclosed herein are oligosaccharide preparations containing anhydrosugar components and/or sugar dehydration product components that exhibit complex functional modulation of a microbial community, such as the animal gut microbiome. Oligosaccharide formulations provide benefits for regulating fermentable carbon utilization by microflora and direct metabolic flux to beneficial species, thereby providing microbiome-mediated improvements in health or nutrition.

Indigestible carbohydrates can act as prebiotics, providing a fermentable carbon source for the microbial community. For example, diets rich in soluble plant fiber have been identified by their ability to nourish the gut microflora. In addition, bifidogenic prebiotics support the growth of bifidobacteria (for example, representatives of the genus Bifidobacterium), and lactogenic prebiotics support the growth of Lactobacillus species.

Prebiotic fibers can be fermented into beneficial chemicals such as short-chain fatty acids (SCFAs). Prebiotic fibers include resistant starches; cellulose; pectins such as rhamnogalactans, arabinogalactans, arabinans; hemicelluloses such as arabinoxylans, xyloglucans, glucomannans, galactomannans; xylans such as corn cob oligosaccharides; b-glucans such as cereal b-glucans, yeast b-glucans, bacterial b-glucans; polyfructans such as inulin and levan; and gums such as alginate. Inulin is a common bifidogenic prebiotic fiber.

In other cases, prebiotics act by interfering with the ability of pathogenic bacteria to establish themselves and thus infect the host, through counteraction mechanisms such as competitive binding of cell surface receptors. Some galactooligosaccharides effectively inhibit the adhesion of various enteropathogenic organisms, such as Escherichia species.

Prebiotics are typically administered to the animal host by inclusion in the diet, where they exhibit a dose-dependent response (at least up to the saturation threshold). For example, administration of a higher dose of a bifidogenic prebiotic such as inulin tends to increase the population of Bifidobacterium species more. Higher doses of inulin correspond to higher production of SCFAs through fermentation. This is because the prebiotic is a source of metabolic carbon, and more carbon is converted into a more fermentable product. Similarly, the use of a higher dose of anti-adhesion prebiotic provides the likelihood of competitive binding of surface receptor sites.

Certain types of carbohydrates containing modified monomeric subunits may influence the way microbial systems utilize other carbohydrates otherwise available to them as a source of prebiotics. For example, such types of carbohydrates may be modified types of carbohydrates that modulate the bacterial starch utilization system (SUS), that is, proteins responsible for cell surface recognition, glycosidic degradation and import of starch metabolites.

Carbohydrate compositions capable of complex modulation of the animal microbiota can be used as feed additives that improve animal health and nutrition through effects on the animal microbiota. For example, modulation of butyrate production by gut microbiota provides health benefits to the animal by promoting healthy intestinal mucosa, barrier function, and providing anti-inflammatory effects. Modulation of propionic acid production affects the metabolic energy extracted from the animal's diet by increasing gluconeogenesis. Suitable microbial communities include, for example, the ileal, jejunal, cecal and/or fecal microbiota of poultry, pigs, dogs, cats, horses, or the ruminant microbiota of cattle, cows, sheep, etc. Other microbial communities include skin microflora, nasal microflora, etc.

In addition, the oligosaccharide preparations described herein have the advantage that they can be selectively analyzed and quantified in a complex nutritional composition such as animal feed due to the presence of anhydrosubunits. It is commercially useful to analyze the presence and/or concentration of feed additives such as oligosaccharide preparations. Such analysis may be performed for quality control purposes to determine whether the supplement has been properly mixed with the base nutritional composition to produce a final nutritional composition containing the supplement at the intended dose or inclusion level.

However, the nutritional compositions themselves contain a large amount and variety of carbohydrate structures (eg, starch, plant fibers and pectins). It is therefore particularly difficult to distinguish small quantities of oligosaccharide-based feed additives from the vast quantities of other carbohydrates present as the basis of the nutritional composition. Thus, the oligosaccharide preparation described herein provides a means of distinguishing itself from other sources of carbohydrates in a nutritional composition through anhydrosubunits.

B. Distribution by degree of polymerization (DP)

In some embodiments, the oligosaccharide preparation contains at least n oligosaccharide fractions, each of which has a different degree of polymerization selected from 1 to n (fractions DP1-DPn). In some embodiments, the oligosaccharide preparation contains n oligosaccharide fractions, where each fraction has a different degree of polymerization selected from 1 to n (fractions DP1 to DPn). For example, in some embodiments, the DP1 fraction contains one or more monosaccharides and/or one or more anhydromonosaccharides. As another example, in some embodiments, the DP1 fraction includes glucose, galactose, fructose, 1,6-anhydro-β-O-glucofuranose, 1,6-anhydro-β-D-glucopyranose, or any combination thereof. As another example, in some embodiments, the DP2 fraction contains one or more regular disaccharides and one or more anhydrous subunit-containing disaccharides. In some embodiments, the DP2 fraction contains lactose.

In some embodiments, n is at least 2, at least 3, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31 , at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41 , at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51 , at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61 , at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71 , at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81 , at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, at least 88, at least 89, at least 90, at least 91 , at least 92, at least 93, at least 94, at least 95, at least 96, at least

- 9 044359

97, at least 98, at least 99, or at least 100. In some embodiments, n is 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,

30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,

60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,

90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100. In some embodiments, n is less than 10, less than 11, less than 12, less than 13, less than 14, less than 15, less than 16, less 17, less than 18, less than 19, less than 20, less than 21, less than 22, less than 23, less than 24, less than 25, less than 26, less than 27, less than 28, less than 29, less than 30, less than 31, less than 32, less than 33, less than 34, less than 35, less than 36, less than 37, less than 38, less than 39, less than 40, less than 41, less than 42, less than 43, less than 44, less than 45, less than 46, less than 47, less than 48, less than 49, less than 50 , less than 51, less than 52, less than 53, less than 54, less than 55, less than 56, less than 57, less than 58, less than 59, less than 60, less than 61, less than 62, less than 63, less than 64, less than 65, less than 66, less 67, less than 68, less than 69, less than 70, less than 71, less than 72, less than 73, less than 74, less than 75, less than 76, less than 77, less than 78, less than 79, less than 80, less than 81, less than 82, less than 83, less than 84, less than 85, less than 86, less than 87, less than 88, less than 89, less than 90, less than 91, less than 92, less than 93, less than 94, less than 95, less than 96, less than 97, less than 98, less than 99 or less than 100 In some embodiments, n is from 2 to 100, from 5 to 90, from 10 to 90, from 10 to 80, from 10 to 70, from 10 to 60, from 10 to 50, from 10 to 40, from 10 to 30, 15 to 60, 15 to 50, 15 to 45, 15 to 40, 15 to 35 or 15 to 30.

The distribution of the degree of polymerization of an oligosaccharide preparation can be determined by any suitable analytical method and equipment, including the end group method, osmotic pressure (osmometry), ultracentrifugation, viscosity measurement, light scattering method, size exclusion chromatography (SEC), SEC-MALLS, flow-through force field fractionation ( PFP), asymmetric force field flow fractionation (A4F), high performance liquid chromatography (HPLC) and mass spectrometry (MS), but not limited to. For example, the distribution of the degree of polymerization can be determined and/or detected using mass spectrometry, such as matrix-assisted laser desorption/ionization mass spectrometry (MALDI)-MS, liquid chromatography (LC)-MS, or gas chromatography (GC)-MS. In another example, the distribution of the degree of polymerization can be determined and/or detected using SEC, such as gel permeation chromatography (GPC). As another example, the distribution of the degree of polymerization can be determined and/or detected using HPLC, PFP or A4F. In some embodiments, the degree of polymerization distribution is determined and/or detected using MALDI-MS. In some embodiments, the degree of polymerization distribution is determined and/or detected using GC-MS or LC-MS. In some embodiments, the degree of polymerization distribution is determined and/or detected using SEC. In some embodiments, the degree of polymerization distribution is determined and/or detected by HPLC. In some embodiments, the polymerization degree distribution is determined and/or detected using a combination of analytical tools such as MALDI-MS and SEC. In some embodiments, the degree of polymerization of an oligosaccharide drug can be determined based on its molecular weight and molecular weight distribution. For example, FIG. 2 shows a MALDI-MS spectrum that illustrates the degrees of polymerization of the various fractions and the presence of oligosaccharides containing anhydrous subunits (peak offset -18 g/mol MW) in all observed fractions.

In some embodiments, the relative abundance of oligosaccharides in most fractions decreases monotonically with the degree of polymerization. In some embodiments, the relative abundance of oligosaccharides of less than 6, less than 5, less than 4, less than 3, or less than 2 fractions of the oligosaccharide preparation does not decrease monotonically with its degree of polymerization.

In some embodiments, the relative abundance of oligosaccharides is at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least In at least 45 or at least 50 fractions, DP decreases monotonically with the degree of polymerization. In some embodiments, the relative abundance of oligosaccharides is at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least in at least 45 or at least 50 successive fractions, DP decreases monotonically with the degree of polymerization. In some embodiments, the relative abundance of oligosaccharides in at least 5, at least 10, at least 20, or at least 30 DP fractions decreases monotonically with degree of polymerization. In some embodiments, the relative abundance of oligosaccharides in at least 5, at least 10, at least 20, or at least 30 successive DP fractions decreases monotonically with degree of polymerization.

In some embodiments, the relative abundance of oligosaccharides in each of the n fractions decreases monotonically with the degree of polymerization. For example, FIG. 15 is an example of a DP distribution where the relative abundance of oligosaccharides in each of the n fractions decreases monotonically with DP. For example, in some embodiments, only the relative abundance of oligosaccharides in the DP3 fraction does not decrease monotonically with degree

- 10 044359 polymerization, i.e. the relative abundance of oligosaccharides in the DP3 fraction is lower than the relative abundance of oligosaccharides in the DP4 fraction. In some embodiments, the relative abundance of oligosaccharides in the DP2 fraction is lower than the relative abundance of oligosaccharides in the DP3 fraction. For example, FIG. 16 illustrates the distribution according to the degree of polymerization, in which the relative abundance of oligosaccharides in the DP2 fraction does not decrease monotonically with the degree of polymerization.

In some embodiments, the oligosaccharide preparation described herein has a DP1 fraction content of about 1 to 50%, about 1 to 40%, about 1 to 35%, about 1 to 30%, about 1 to 25%, about from 1 to 20%, from about 1 to 15%, from about 5 to 50%, from about 5 to 40%, from about 5 to 35%, from about 5 to 30%, from about 5 to 25%, from about 5 to 20%, about 5 to 15%, about 10 to 50%, about 10 to 40%, about 10 to 35%, about 10 to 30%, about 10 to 25%, about 10 up to 20% or about 10 to 15% by weight or relative abundance. In some embodiments, the oligosaccharide preparation has a DP1 fraction content of about 10 to 35%, about 10 to 20%, or about 10 to 15% by weight or relative abundance. In some embodiments, the content of the DP1 fraction is determined by MALDI-MS. In some embodiments, the DP1 fraction is determined by HPLC. In some embodiments, the content of the DP1 fraction is determined using LC-MS/MS or GC-MS.

In some embodiments, the oligosaccharide preparation described herein has a DP2 fraction content of about 1 to 35%, about 1 to 30%, about 1 to 25%, about 1 to 20%, about 1 to 15%, about 1 to 10%, about 5 to 30%, about 5 to 25%, about 5 to 20%, about 5 to 15%, or about 5 to 10% by weight or relative abundance. In some embodiments, the oligosaccharide preparation has a DP2 fraction content of about 5 to 25%, about 5 to 20%, about 5 to 15%, or about 5 to 10% by weight or relative abundance. In some embodiments, the DP2 fraction is determined by MALDI-MS. In some embodiments, the DP2 fraction is determined by HPLC. In some embodiments, the DP2 fraction is determined using LC-MS/MS or GC-MS.

In some embodiments, the oligosaccharide preparation described herein has a DP3 fraction content of about 1 to 30%, about 1 to 25%, about 1 to 20%, about 1 to 15%, about 1 to 10%, about 5 up to 30%, about 5 to 25%, about 5 to 20%, about 5 to 15%, or about 5 to 10% by weight or relative abundance. In some embodiments, the oligosaccharide preparation has a DP3 fraction content of about 1 to 15%, about 1 to 10%, about 5 to 15%, or about 5 to 10% by weight or relative abundance. In some embodiments, the content of the DP3 fraction is determined using MALDI-MS. In some embodiments, the DP3 fraction is determined using HPLC. In some embodiments, the DP3 fraction is determined using LC-MS/MS or GC-MS.

In some embodiments, the oligosaccharide preparation described herein has a DP4 fraction content of about 0.1 to 20%, about 0.1 to 15%, about 0.1 to 10%, about 0.1 to 5%, about 1 to 20%, about 1 to 15%, about 1 to 10%, or about 1 to 5% by weight or relative abundance. In some embodiments, the oligosaccharide preparation has a DP4 fraction content of about 1 to 15%, about 1 to 10%, or about 1 to 5% by weight or relative abundance. In some embodiments, the oligosaccharide preparation described herein has a DP5 fraction content of about 0.1 to 15%, about 0.1 to 10%, about 0.1 to 5%, about 1 to 15%, about 1 to 10% or about 1 to 5% by weight or relative abundance. In some embodiments, the oligosaccharide preparation has a DP5 fraction content of about 1 to 10%, or about 1 to 5% by weight or relative abundance. In some embodiments, the content of the DP4 and/or DP5 fraction is determined using MALDIMS. In some embodiments, the content of the DP4 and/or DP5 fraction is determined using HPLC. In some embodiments, the content of the DP4 and/or DP5 fraction is determined using LC-MS/MS or GC-MS.

In some embodiments, the ratio of fraction DP2 to fraction DP1 in the oligosaccharide preparation is about 0.01 to 0.8, about 0.02 to 0.7, about 0.02 to 0.6, about 0.02 to 0.5, about 0.02 to 0.4, about 0.02 to 0.3, about 0.02 to 0.2, about 0.1 to 0.6, about 0.1 to 0.5, about 0.1 to 0.4, or about 0.1 to 0.3 by weight or relative abundance. In some embodiments, the ratio of the DP2 fraction to the DP1 fraction in the oligosaccharide preparation is from about 0.02 to 0.4 by weight or relative abundance.

In some embodiments, the ratio of fraction DP3 to fraction DP2 in the oligosaccharide preparation is about 0.01 to 0.7, about 0.01 to 0.6, about 0.01 to 0.5, about 0.01 to 0.4, about 0.01 to 0.3, or about 0.01 to 0.2 by weight or relative abundance. In some embodiments, the ratio of the DP3 fraction to the DP2 fraction in the oligosaccharide preparation is from about 0.01 to 0.3 by weight or relative abundance.

In some embodiments, the combined content of the DP1 and DP2 fractions in the oligosaccharide preparation is less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% by weight or relative abundance. In some embodiments, the combined content of the DP1 and DP2 fractions in the oligosaccharide preparation is less than 50%, less than 30%, or less than 10% by weight or relative abundance.

In some embodiments, the oligosaccharide preparation described herein has an average DP value in the range of 2 to 10. In some embodiments, the oligosaccharide preparation has an average DP value of about 2 to 8, about 2 to 5, or about 2 to 4 In some embodiments, the oligosaccharide preparation has an average DP value of about 3.5. The average DP value can be determined using SEC or elemental analysis.

C. Level of anhydrosubunits

In some embodiments, each of the n oligosaccharide fractions in an oligosaccharide preparation described herein independently includes a level of anhydrosubunits. For example, in some embodiments, fraction DP1 contains about 10% anhydrosubunit-containing oligosaccharides by relative abundance, and fraction DP2 contains about 15% anhydrosubunit-containing oligosaccharides by relative abundance. In another example, in some embodiments, each fraction of DP1, DP2, and DP3 contains about 5%, about 10%, and about 2% anhydrosubunit-containing oligosaccharides, by relative abundance, respectively. In some embodiments, two or more oligosaccharide fractions contain similar levels of anhydrosubunit-containing oligosaccharides. For example, in some embodiments, the DP1 and DP3 fractions each contain approximately 5% anhydrosubunit-containing oligosaccharides, by relative abundance.

In some embodiments, each of fractions 1 to n in an oligosaccharide preparation described herein independently comprises from about 0.1 to 15% anhydrous subunit-containing oligosaccharides, as measured by mass spectrometry, LC-MS/MS, or GC-containing oligosaccharides. MS. In some embodiments, each of fractions 1 to n in the oligosaccharide preparation independently comprises from about 0.5 to 15% oligosaccharides containing anhydrosubunits, as measured by relative abundance by mass spectrometry, LC-MS/MS, or GC-MS. In some embodiments, LC-MS/MS is used to determine the relative abundance of oligosaccharides in the DP1, DP2 and/or DP3 fractions. In some embodiments, GC-MS or LC-MS/MS is used to determine the relative abundance of oligosaccharides in the DP1, DP2 and/or DP3 fractions. In some embodiments, MALDI-MS is used to determine the relative abundance of oligosaccharides in the DP4 or higher DP fraction. In some embodiments, the relative abundance of a particular fraction is determined by integrating the area under the LC-MS/MS chromatogram peaks that are designated as corresponding to that fraction. In some embodiments, the relative abundance of a particular fraction is calculated by integrating the area under the GC-MS chromatogram peaks that are designated as corresponding to that fraction.

The level of anhydrosubunits can be determined and/or detected by any suitable analytical methods, such as nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, HPLC, PFP, A4F, or any combination thereof. In some embodiments, the level of oligosaccharides containing anhydrosubunits is determined, at least in part, by mass spectrometry, such as MALDI-MS. In some embodiments, the level of anhydrosubunits is determined, at least in part, using NMR. In some embodiments, the level of oligosaccharides containing anhydrosubunits is determined, at least in part, by HPLC. In some embodiments, the level of oligosaccharides containing anhydrosubunits is determined by MALDI-MS, as indicated by the peaks offset at -18 g/mol MW in FIG. 2. In some embodiments, the presence and type of anhydro subunit species is determined and/or detected by NMR, as illustrated in Example 11 of FIG. 3 and 4. In some embodiments, the relative abundance of oligosaccharides containing anhydrosubunits is determined using MALDI-MS. In some embodiments, the relative abundance of oligosaccharides containing anhydrosubunits is determined using LC-MS/MS, as shown in FIG. 26A-26C, 27A-27C, 28A-28C and 29A-29C. In some embodiments, the relative abundance of oligosaccharides containing anhydrosubunits is determined using GC-MS, as shown in FIG. 30A-30B, 31A-31B, 32A-32B and 33A-33B.

In some embodiments, at least one fraction of an oligosaccharide preparation described herein comprises less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7% , less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of oligosaccharides containing anhydrosubunits, by relative abundance. In some embodiments, at least one fraction of an oligosaccharide preparation described herein comprises less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, or less than 2 % of oligosaccharides containing anhydrosubunits, by relative abundance. In other embodiments, at least one fraction of an oligosaccharide preparation described herein comprises greater than 0.5%, greater than 0.8%, greater than 1%, greater than 2%, greater than 3%, greater than 4%, greater than 5%, greater than 6 %, more than 7%, more than 8%, more than 9%, more than 10%, more than 11%, more than 12%, more than 13%, more than 14%, more than 15%, more than 16%, more than 17%, more than 18%, more than 19%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, or more than 80% oligosaccharides containing anhydrosubunits, by relative abundance. In other embodiments, at least one fraction of an oligosaccharide preparation described herein comprises greater than 20%, greater than 21%, greater than 22%, greater than 23%, greater than 24%, greater than 25%, greater than 26%, greater than 27%, greater than 28 %, more than 29% or more than 30% of oligosaccharides containing anhydrosubunits, by relative abundance. In some embodiments, at least one fraction (such as DP1, DP2, and/or DP3) of the oligosaccharide drug contains about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0. 5%, approximately 0.6%, approximately 0.7%, approximately 0.8%, approximately 0.9%, approximately 1%, approximately 1.5%, approximately 2%, approximately 3%, approximately 4%, approximately 5%, approximately 6%, approximately 7%, approximately 8%, approximately 9%, approximately 10%, approximately 11%, approximately 12%, approximately 13%, approximately 14%, approximately 15%, approximately 16%, approximately 17% , about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, or about 30% of anhydrosubunit-containing oligosaccharides, by relative abundance. In some embodiments, at least one fraction (such as DP1, DP2, and/or DP3) of the oligosaccharide drug contains about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0. 5%, approximately 0.6%, approximately 0.7%, approximately 0.8%, approximately 0.9%, approximately 1%, approximately 2%, approximately 3%, approximately 4%, approximately 5%, approximately 6% , about 7%, about 8%, about 9%, or about 10% of oligosaccharides containing anhydrosubunits, by relative abundance. In some embodiments, at least one fraction (such as DP1, DP2, and/or DP3) of the oligosaccharide preparation contains from about 0.1 to 90%, about 0.5 to 90%, about 0.5 to 80%, about 0.5 to 70%, about 0.5 to 60%, about 0.5 to 50%, about 0.5 to 40%, about 0.5 to 30%, about 0.5 up to 20%, about 0.5 to 10%, about 0.5 to 9%, about 0.5 to 8%, about 0.5 to 7%, about 0.5 to 6% about 0.5 to 5%, about 0.5 to 4%, about 0.5 to 3%, about 0.5 to 2%, about 1 to 10%, about 2 to 9%, about 2 to 8%, about 2 to 7%, about 2 to 6%, about 2 to 5%, about 2 to 4%, about 2 to 3%, or about 5 to 10% oligosaccharides containing anhydrosubunits , by relative prevalence. In some embodiments, each of the oligosaccharide preparation fractions DP1 and DP2 independently comprises anhydrosubunit-containing oligosaccharides ranging from about 0.1%; 0.5%; 0.6%; 0.7%; 0.8%; 0.9%; 1%; 1.1%; 1.2%; 1.3%; 1.4% or 1.5% to about 8%, 9%, 10%, 11%, 12%, or 15% by relative abundance measured by mass spectrometry, LC-MS/MS, or GC-MS. In some embodiments, the DP1 and DP2 fractions each independently comprise from about 0.5% to 15% anhydrosubunit-containing oligosaccharides, as measured by relative abundance by mass spectrometry or LC-MS/MS or GC-MS.

In some embodiments, each fraction of an oligosaccharide preparation described herein comprises less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 19%, less than 18%, less 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5% , less than 4%, less than 3%, less than 2%, or less than 1% of oligosaccharides containing anhydrosubunits, by relative abundance. In some embodiments, each fraction of an oligosaccharide preparation described herein contains less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% anhydrosubunit-containing oligosaccharides, by relative abundance. In other embodiments, each fraction of the oligosaccharide preparation described herein contains greater than 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% anhydrosubunit-containing oligosaccharides, by relative abundance. In other embodiments, each fraction of the oligosaccharide preparation described herein contains greater than 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% anhydrosaccharide-containing oligosaccharides , by relative prevalence. In some embodiments, each fraction of an oligosaccharide preparation described herein comprises about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, approximately 0.8%, approximately 0.9%, approximately 1%, approximately 1.5%, approximately 2%, approximately 3%, at

- 13 044359 approximately 4%, approximately 5%, approximately 6%, approximately 7%, approximately 8%, approximately 9%, approximately 10%, approximately 11%, approximately 12%, approximately 13%, approximately 14%, approximately 15% , about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25% or about 30% oligosaccharides containing anhydrosubunits, according to relative prevalence. In some embodiments, each fraction of an oligosaccharide preparation described herein comprises about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, approximately 0.8%, approximately 0.9%, approximately 1%, approximately 2%, approximately 3%, approximately 4%, approximately 5%, approximately 6%, approximately 7%, approximately 8%, approximately 9% or approximately 10% of oligosaccharides containing anhydrosubunits, by relative abundance. In some embodiments, each fraction of an oligosaccharide preparation described herein comprises from about 0.1 to 90%, about 0.1 to 15%, about 0.5 to 90%, about 0.5 to 80%, about from 0.5 to 70%, from about 0.5 to 60%, from about 0.5 to 50%, from about 0.5 to 40%, from about 0.5 to 30%, from about 0.5 to 20%, about 0.5 to 10%, about 0.5 to 9%, about 0.5 to 8%, about 0.5 to 7%, about 0.5 to 6%, about 0.5 to 5%, about 0.5 to 4%, about 0.5 to 3%, about 0.5 to 2%, about 2 to 9%, about 2 to 8%, about 2 to 7%, about 2 to 6%, about 2 to 5%, about 2 to 4%, about 2 to 3%, or about 5 to 10% anhydrosubunit-containing oligosaccharides, by relative abundance.

In some embodiments, the oligosaccharide preparation described herein contains less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 19%, less than 18%, less than 17% , less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less 4%, less than 3%, less than 2%, or less than 1% of oligosaccharides containing anhydrosubunits, by relative abundance. In some embodiments, the oligosaccharide preparation contains less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% anhydrosubunit-containing oligosaccharides, by relative abundance. In other embodiments, the oligosaccharide preparation contains greater than 0.5%, greater than 0.8%, greater than 1%, greater than 2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%, greater than 7%, greater than 8% , more than 9%, more than 10%, more than 11%, more than 12%, more than 13%, more than 14%, more than 15%, more than 16%, more than 17%, more than 18%, more than 19%, more than 20%, more 30%, more than 40%, more than 50%, more than 60%, more than 70%, or more than 80% of oligosaccharides containing anhydrosubunits, by relative abundance. In other embodiments, the oligosaccharide preparation contains greater than 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% anhydrosubunit-containing oligosaccharides, by relative abundance. In some embodiments, the oligosaccharide preparation contains about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, approximately 0.9%, approximately 1%, approximately 2%, approximately 3%, approximately 4%, approximately 5%, approximately 6%, approximately 7%, approximately 8%, approximately 9%, approximately 10% , approximately 11%, approximately 12%, approximately 13%, approximately 14%, approximately 15%, approximately 16%, approximately 17%, approximately 18%, approximately 19%, approximately 20%, approximately 21%, approximately 22%, approximately 23%, about 24%, about 25%, or about 30% of oligosaccharides containing anhydrosubunits, by relative abundance. In some embodiments, the oligosaccharide preparation contains about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, approximately 0.9%, approximately 1%, approximately 1.5%, approximately 2%, approximately 3%, approximately 4%, approximately 5%, approximately 6%, approximately 7%, approximately 8%, approximately 9% or approximately 10% of oligosaccharides containing anhydrosubunits, by relative abundance. In some embodiments, the oligosaccharide preparation comprises about 0.1 to 90%, about 0.1 to 15%, about 0.5 to 90%, about 0.5 to 80%, about 0.5 to 70 %, about 0.5 to 60%, about 0.5 to 50%, about 0.5 to 40%, about 0.5 to 30%, about 0.5 to 20%, about 0 .5 to 10%, about 0.5 to 9%, about 0.5 to 8%, about 0.5 to 7%, about 0.5 to 6%, about 0.5 to 5% , about 0.5 to 4%, about 0.5 to 3%, about 0.5 to 2%, about 2 to 9%, about 2 to 8%, about 2 to 7%, about 2 to 6%, about 2 to 5%, about 2 to 4%, about 2 to 3%, or about 5 to 10% anhydrous subunit-containing oligosaccharides, by relative abundance.

In some embodiments, the DP1 fraction of an oligosaccharide drug described herein contains less than 30%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1% oligosaccharides containing anhydrosubunits , by relative prevalence. In some embodiments, the DP1 fraction of an oligosaccharide drug described herein includes more than 0.1%, more than 0.5%, more than 0.8%, more than 1%, more than 1.5%, more than 2%, more than 3%, more 4%, more than 5%, more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 11%, more than 12%, more than 13%, more than 14% or more than 15% oligosaccharides containing anhydrosubunits , by relative prevalence. In some embodiments, the DP1 fraction of an oligosaccharide drug described herein contains approximately

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0.5%, approximately 1%, approximately 2%, approximately 3%, approximately 4%, approximately 5%, approximately 6%, approximately 7%, approximately 8%, approximately 9%, approximately 10%, approximately 11%, approximately 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% anhydrous subunit-containing oligosaccharides, by relative abundance. In some embodiments, the DP1 fraction of an oligosaccharide drug described herein contains from about 0.1 to 15%, about 0.1 to 20%, about 0.5 to 20%, about 0.5 to 10%, about from 0.5 to 15%, from about 1 to 20%, from about 1 to 15%, from about 1 to 10%, from about 2 to 14%, from about 3 to 13%, from about 4 to 12%, about 5 to 11%, about 5 to 10%, about 6 to 9%, or about 7 to 8% of the anhydrosubunit-containing oligosaccharides, by relative abundance, or any range therebetween. In some embodiments, the DP1 fraction of an oligosaccharide preparation described herein comprises from about 0.5 to 10% anhydrosaccharide-containing oligosaccharides, by relative abundance. In some embodiments, the relative abundance of oligosaccharides containing anhydrosubunits is determined using mass spectrometry, such as MALDIMS. In some embodiments, the relative abundance of oligosaccharides containing anhydrosubunits is determined using LC-MS/MS. In some embodiments, the relative abundance of oligosaccharides containing anhydrosubunits is determined using GC-MS.

In some embodiments, the DP2 fraction of an oligosaccharide drug described herein contains less than 30%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1% oligosaccharides containing anhydrosubunits , by relative prevalence. In some embodiments, the DP2 fraction of an oligosaccharide drug described herein includes more than 0.1%, more than 0.5%, more than 0.8%, more than 1%, more than 1.5%, more than 2%, more than 3%, more 4%, more than 5%, more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 11%, more than 12%, more than 13%, more than 14% or more than 15% oligosaccharides containing anhydrosubunits , by relative prevalence. In some embodiments, the DP2 fraction of an oligosaccharide drug described herein contains about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8% , about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19% or about 20% oligosaccharides, containing anhydrosubunits, according to relative abundance. In some embodiments, the DP2 fraction of an oligosaccharide drug described herein contains from about 0.1 to 15%, about 0.1 to 20%, about 0.5 to 20%, about 0.5 to 10%, about from 0.5 to 15%, from about 1 to 20%, from about 1 to 15%, from about 1 to 10%, from about 2 to 14%, from about 3 to 13%, from about 4 to 12%, about 5 to 11%, about 5 to 10%, about 6 to 9%, or about 7 to 8% anhydrosaccharide-containing oligosaccharides, by relative abundance, or any range therebetween. In some embodiments, the DP2 fraction of an oligosaccharide preparation described herein comprises about 5 to 10% anhydrosubunit-containing oligosaccharides by relative abundance. In some embodiments, the relative abundance of oligosaccharides containing anhydrosubunits is determined using mass spectrometry, such as MALDIMS. In some embodiments, the relative abundance of oligosaccharides containing anhydrosubunits is determined using LC-MS/MS. In some embodiments, the relative abundance of oligosaccharides containing anhydrosubunits is determined using GC-MS.

In some embodiments, the DP3 fraction of an oligosaccharide drug described herein contains less than 30%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1% oligosaccharides containing anhydrosubunits , by relative prevalence. In some embodiments, the DP3 fraction of an oligosaccharide drug described herein includes more than 0.1%, more than 0.5%, more than 0.8%, more than 1%, more than 1.5%, more than 2%, more than 3%, more 4%, more than 5%, more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 11%, more than 12%, more than 13%, more than 14% or more than 15% oligosaccharides containing anhydrosubunits , by relative prevalence. In some embodiments, the DP3 fraction of an oligosaccharide drug described herein contains about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8% , about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16% about 17%, about 18%, about 19% or about 20% oligosaccharides containing anhydrosubunits, by relative abundance. In some embodiments, the DP3 fraction of an oligosaccharide drug described herein contains from about 0.1 to 15%, about 0.1 to 20%, about 0.5 to 20%, about 0.5 to

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10%, about 0.5 to 15%, about 1 to 20%, about 1 to 15%, about 1 to 10%, about 2 to 14%, about 3 to 13%, about 4 to 12%, about 5 to 11%, about 5 to 10%, about 6 to 9%, or about 7 to 8% anhydrosubunit-containing oligosaccharides, by relative abundance, or any ranges therebetween. In some embodiments, the DP3 fraction of an oligosaccharide preparation described herein contains from about 0.5 to 10% anhydrosubunit-containing oligosaccharides, by relative abundance. In some embodiments, the relative abundance of oligosaccharides containing anhydrosubunits is determined using mass spectrometry, such as MALDIMS. In some embodiments, the relative abundance of oligosaccharides containing anhydrosubunits is determined using LC-MS/MS. In some embodiments, the relative abundance of oligosaccharides containing anhydrosubunits is determined using GC-MS.

In some embodiments, the anhydrosubunit-containing oligosaccharide includes one or more anhydrosubunits. For example, the DP1 anhydrosubunit-containing oligosaccharide includes one anhydrosubunit. In some embodiments, the DPn anhydrosubunit-containing oligosaccharide may contain from 1 to n anhydrosubunits. For example, in some embodiments, the DP2 anhydrosubunit-containing oligosaccharide includes one or two anhydrosubunits. In some embodiments, each oligosaccharide in the oligosaccharide preparation independently contains zero, one, or two anhydrosubunits. In some embodiments, greater than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 30% oligosaccharides , containing anhydrosubunits, have only one anhydrosubunit. In some embodiments, more than 99%, 95%, 90%, 85%, or 80% of the anhydrosubunit-containing oligosaccharides contain only one anhydrosubunit.

In some embodiments, the one or more oligosaccharides in the oligosaccharide preparation or in each fraction of the oligosaccharide preparation contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 anhydrosubunits, each of which is linked through a glycosidic bond, where the glycosidic bond , binding each anhydrosubunit was selected independently. In some embodiments, one or more oligosaccharides in the oligosaccharide preparation in or each fraction of the oligosaccharide preparation contain 1, 2, or 3 anhydrosubunits, each of which is linked by a glycosidic bond, wherein the glycosidic bond linking each anhydrosubunit is independently selected. In some embodiments, more than 50%, 60%, 70%, 80%, 90%, or 99% of the oligosaccharides in the oligosaccharide preparation or in each fraction contain 1, 2, or 3 anhydrosubunits, each of which is linked by a glycosidic bond, wherein the glycosidic bond linking each anhydrosubunit, selected independently. In some embodiments, one or more oligosaccharides in the oligosaccharide preparation or in each fraction contain 1 anhydrosubunit linked by a glycosidic linkage. In some embodiments, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or more than 99% of the oligosaccharides in the oligosaccharide preparation or in each fraction contain 1 anhydrosubunit linked through a glycosidic bond.

D. Types of anhydrosubunits

In some embodiments, the oligosaccharide preparation contains various types of anhydrosubunits. In some embodiments, exemplary oligosaccharides containing anhydrosubunits are illustrated in FIG. 35, 23 and 24. In some embodiments, the oligosaccharide preparation contains one or more anhydrosaccharide subunits, which are products of thermal dehydration of monosaccharides, that is, anhydrosaccharide subunits. In some embodiments, the oligosaccharide preparation contains one or more anhydrosubunits that are products of the reversible thermal dehydration of monosaccharides.

It should be understood that an anhydromonosaccharide (or anhydromonosaccharide subunit) refers to one or more types of thermal dehydration products of a monosaccharide. For example, in some embodiments, anhydroglucose refers to 1,6-anhydro-O-glucopyranose (levoglucosan) or 1,6-anhydro-O-glucofuranose. In some embodiments, the plurality of anhydroglucoses refers to a plurality of 1,6-anhydro-β-O-glucopyranoses (levoglucosans), a plurality of 1,6anhydro-β-O-glucofuranoses, a variety of other thermal dehydration products of glucose, or any combination thereof. Likewise, in some embodiments, the term anhydrogalactose plurality refers to the plurality of any thermal dehydration products of galactose or any combination thereof.

In some embodiments, the oligosaccharide preparation as described herein contains one or more subunits of anhydroglucose, anhydrogalactose, anhydromannose, anhydroallose, anhydroaltrose, anhydrogulose, anhydroindose, anhydrothalose, anhydrofructose, anhydroribose, anhydroarabinose, anhydrorhamnose, anhydrolyxose, anhydroxyloses, or any combination of these subunits. In some embodiments, the oligosaccharide preparation contains one or more anhydroglucose, anhydrogalactose, anhydromannose, or anhydrofructose subunits. In some embodiments, the oligosaccharide preparation as described herein contains one or more of 1,6-anhydro-3-O-b-O-glucopyranosyl-b-O-glucopyranose, 1,6-anhydro-3-O-a- O-glucopyranosyl-in-O-glucopyranoses, 1,6-anhydro-2-O-in-O-glucopyranosyl-in-O-glucopyranoses, 1,6-anhydro-2-O-a-O-glucopyranosyl-in-O- glucopyranose, 1,6-anhydro-O-cellobiose (cellobiosan), 1,6-anhydro-β-D-cellotriose (cellotriosan), 1,6-anhydro-O-cellotetraose (cellotetraosan), 1,6 -anhydro-O-cellopentaose (cellopentaosane) and 1,6-anhydro-O-maltose (maltosan).

In some embodiments, the oligosaccharide preparation contains one or more 1,6-anhydro-β-O-glucofuranose subunits. In some embodiments, the oligosaccharide preparation contains one or more 1,6-anhydro-β-D-glucopyranose (levoglucosan) subunits. For example, FIG. 35 illustrates two DP1 oligosaccharides containing anhydrosubunits (levoglucosan and 1,6-anhydro-β-O-glucofuranose) and a DP2 oligosaccharide containing anhydrosubunits (anhydrocellobiose).

The presence and level of anhydrosubunit species may vary depending on the feed sugars used to produce the oligosaccharide. For example, in some embodiments, gluco-oligosaccharides contain anhydroglucose subunits, galacto-oligosaccharides contain anhydrogalactose subunits, and glucogalacto-oligosaccharides contain anhydroglucose and anhydrogalactose subunits.

In some embodiments, the oligosaccharide preparation contains both 1,6-anhydro-β-Oglucofuranose and 1,6-anhydro-β-D-glucopyranose anhydrosubunits. In some embodiments, at least 0.1%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% of the anhydrosubunits is selected from group consisting of 1,6-anhydro-β-O-glucofuranose and 1,6anhydro-β-O-glucopyranose. In some embodiments, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the anhydro subunits are 1,6-anhydro-b anhydro subunits -O-glucofuranoses. In some embodiments, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, or 60% of the anhydro subunits are 1,6-anhydro-glucopyranose.

In some embodiments, the ratio of 1,6-anhydro-O-glucofuranose to 1,6-anhydro-O-glucopyranose is about 10:1 to 1:10, 9:1 to 1:10, 8:1 to 1:10, from 7:1 to 1:10, from 6:1 to 1:10, from 5:1 to 1:10, from 4:1 to 1:10, from 3:1 to 1:10, from 2:1 to 1:10, from 10:1 to 1:9, from 10:1 to 1:8, from 10:1 to 1:7, from 10:1 to 1:6, from 10:1 to 1:5, 10:1 to 1:4, 10:1 to 1:3, 10:1 to 1:2 or 1:1 to 3:1 in the preparation. In some embodiments, the ratio of 1,6-anhydro-O-glucofuranose to 1,6anhydro-O-glucopyranose is about 10:1, 9:1, 8:1, 7:1, 6:1, 5: 1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:8, 1:9 or 1:10 in the preparation. In some embodiments, the ratio of 1,6-anhydro-β-O-glucofuranose to 1,6-anhydro-β-O-glucopyranose in the formulation is approximately 2:1.

In some embodiments, the ratio of 1,6-anhydro-O-glucofuranose to 1,6-anhydro-O-glucopyranose is about 10:1 to 1:10, 9:1 to 1:10, 8:1 to 1:10, from 7:1 to 1:10, from 6:1 to 1:10, from 5:1 to 1:10, from 4:1 to 1:10, from 3:1 to 1:10, from 2:1 to 1:10, from 10:1 to 1:9, from 10:1 to 1:8, from 10:1 to 1:7, from 10:1 to 1:6, from 10:1 to 1:5, from 10:1 to 1:4, from 10:1 to 1:3, from 10:1 to 1:2 or from 1:1 to 3:1 in each fraction. In some embodiments, the ratio of 1,6-anhydro-O-glucofuranose to 1,6-anhydro-O-glucopyranose is about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1: 9 or 1:10 in each fraction. In some embodiments, the ratio of 1,6-anhydro-β-O-glucofuranose to 1,6-anhydro-β-O-glucopyranose is approximately 2:1 in each fraction.

In some embodiments, the ratio of 1,6-anhydro-O-glucofuranose to 1,6-anhydro-O-glucopyranose is about 10:1 to 1:10, 9:1 to 1:10, 8:1 to 1:10, from 7:1 to 1:10, from 6:1 to 1:10, from 5:1 to 1:10, from 4:1 to 1:10, from 3:1 to 1:10, from 2:1 to 1:10, from 10:1 to 1:9, from 10:1 to 1:8, from 10:1 to 1:7, from 10:1 to 1:6, from 10:1 to 1:5, 10:1 to 1:4, 10:1 to 1:3, 10:1 to 1:2 or 1:1 to 3:1 in at least one fraction. In some embodiments, the ratio of 1,6-anhydro-O-glucofuranose to 1,6-anhydro-O-glucopyranose is about 10:1, 9:1, 8:1, 7:1, 6:1, 5: 1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10 in at least one faction. In some embodiments, the ratio of 1,6-anhydro-β-O-glucofuranose to 1,6-anhydro-β-O-glucopyranose is about 2:1 in at least one fraction.

In some embodiments, an oligosaccharide preparation described herein includes OP2 oligosaccharides containing anhydrosaccharides. In some embodiments, the oligosaccharide preparation contains anhydrolactose, anhydrosucrose, anhydrocellobiose, or a combination thereof. In some embodiments, the oligosaccharide preparation includes about 2 to 20, 2 to 15, 5 to 20, 5 to 15, or 5 to 10 OP2 species of oligosaccharides containing anhydrosubunits. In some embodiments, the oligosaccharide preparation described herein does not contain cellobiosan or does not contain a detectable level of cellobiosan.

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In some embodiments, an oligosaccharide preparation described herein contains one or more anhydrosubunits that are caramelization products of sugar. In some embodiments, the oligosaccharide preparation contains one or more anhydrosubunits, which are sugar caramelization products selected from the group consisting of methanol; ethanol; furan; methylglyoxal; 2-methylfuran; vinyl acetate; glycolaldehyde; acetic acid; acetol; furfural; 2-furanmethanol; 3-furanmethanol; 2-hydroxycyclopent2-en-1-one; 5-methylfurfural; 2(5H)-furanone; 2-methylcyclopentenolone; levoglucosenone; cyclic hydroxyl lactone; 1,4,3,6-dianhydro-a-O-glucopyranoses; dianhydroglucopyranoses; and 5-hydroxymethylfurfural (5-hmf). In some embodiments, the oligosaccharide preparation contains 5-hmf anhydrosubunits.

In some embodiments, in the oligosaccharide preparation or at least one of the DP fractions, anhydrosubunits that are products of caramelization are less abundant than anhydrosubunits that are products of reversible thermal dehydration of the monosaccharide. In some embodiments, in the oligosaccharide preparation or at least one of the fractions, anhydrosubunits that are products of caramelization are more abundant than anhydrosubunits that are products of reversible thermal dehydration of the monosaccharide. In some embodiments, in the oligosaccharide preparation or at least one of the fractions, the anhydrosubunits that are products of caramelization and the anhydrosubunits that are products of reversible thermal dehydration of the monosaccharide are of similar abundance.

In some embodiments, about 0.01 to 50%, about 0.01 to 40%, about 0.01 to 30%, about 0.01 to 20%, about 0.01 to 10%, about from 0.01 to 5%, from about 0.01 to 4%, from about 0.01 to 3%, from about 0.01 to 2%, from about 0.01 to 1%, from about 0.1 to 0.5%, about 0.1 to 50%, about 0.1 to 40%, about 0.1 to 30%, about 0.1 to 20%, about 0.1 to 10% about from 0.1 to 5%, from about 0.1 to 4%, from about 0.1 to 3%, from about 0.1 to 2%, from about 0.1 to 1%, or from about 0.1 to 0.5% of the anhydrosubunits in the oligosaccharide preparation described herein are caramelization products. In some embodiments, about 0.1 to 5%, about 0.1 to 2%, or about 0.1 to 1% of the anhydrosubunits in the oligosaccharide preparation are caramelization products. In some embodiments, less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9 %, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1% of the anhydrosubunits in the oligosaccharide preparation are caramelization products.

In some embodiments, about 0.01 to 50%, about 0.01 to 40%, about 0.01 to 30%, about 0.01 to 20%, about 0.01 to 10%, about from 0.01 to 5%, from about 0.01 to 4%, from about 0.01 to 3%, from about 0.01 to 2%, from about 0.01 to 1%, from about 0.01 about from 0.1 to 0.5%, from about 0.1 to 50%, from about 0.1 to 40%, from about 0.1 to 30%, from about 0.1 to 20%, from about 0, 1 to 10% about 0.1 to 5%, about 0.1 to 4%, about 0.1 to 3%, about 0.1 to 2%, about 0.1 to 1% or about From 0.1 to 0.5% of the anhydrous subunits in at least one fraction (eg, DP1, DP2 and/or DP3) of the preparation described herein are caramelization products. In some embodiments, about 0.1 to 5%, about 0.1 to 2%, or about 0.1 to 1% of the anhydrosubunits in at least one fraction (e.g., DP1, DP2, and/or DP3) of the drug are caramelization products. In some embodiments, less than 50%, 40%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of anhydrous subunits in at least one fraction of the drug are caramelization products. In some embodiments, less than 20%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5 %, less than 4%, less than 3%, less than 2% or less than 1% of the anhydrous subunits in fractions DP1, DP2 and/or DP3 of the oligosaccharide preparation described herein are caramelization products.

In some embodiments, about 0.01 to 50%, about 0.01 to 40%, about 0.01 to 30%, about 0.01 to 20%, about 0.01 to 10%, about from 0.01 to 5%, from about 0.01 to 4%, from about 0.01 to 3%, from about 0.01 to 2%, from about 0.01 to 1%, from about 0.01 about from 0.1 to 0.5%, from about 0.1 to 50%, from about 0.1 to 40%, from about 0.1 to 30%, from about 0.1 to 20%, from about 0, 1 to 10% about 0.1 to 5%, about 0.1 to 4%, about 0.1 to 3%, about 0.1 to 2%, about 0.1 to 1% or about from 0.1 to 0.5% of the anhydrosubunits in each fraction of the oligosaccharide preparation described in this application are caramelization products. In some embodiments, about 0.1 to 5%, about 0.1 to 2%, or about 0.1 to 1% of the anhydrous subunits in each formulation fraction are caramelization products. In some embodiments, less than 50%, less than 40%, less than 30%, less than 20%, less than 25%, less than 20%, less than 15%, less than 14%, less than 13%, less than more than 12%, less than 11%, less 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than more than 2% or less than 1% of anhydrous subunits in each fraction of the drug are caramelization products.

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In some embodiments, each of the oligosaccharides in an oligosaccharide preparation described herein independently and optionally contains an anhydrosubunit. In some embodiments, two or more independent oligosaccharides contain the same or different anhydrosubunits. In some embodiments, two or more independent oligosaccharides contain different anhydrous subunits. For example, in some embodiments, the oligosaccharide preparation includes a DP1 anhydrosubunit-containing oligosaccharide that includes 1,6-anhydro-β-O-glucopyranose, and a DP2 anhydrosubunit-containing oligosaccharide that includes a 1,6-anhydro-β-D-glucofuranose subunit . In some embodiments, one or more oligosaccharides in an oligosaccharide preparation comprise two or more of the same or different anhydrosubunits.

In some embodiments, in any fraction of the oligosaccharide preparation that has a degree of polymerization of 2 or more (ie, the DP2-DPn fraction), the anhydro subunit may be associated with one or more regular or anhydro subunits. In some embodiments, in the DP2-DPn fractions, at least one anhydrosubunit is associated with one, two, or three other regular or anhydrosubunits. In some embodiments, in the DP2-DPn fractions, at least one anhydrosubunit is associated with one or two regular subunits. In some embodiments, in the DP2-DPn fractions, at least one anhydro subunit is associated with one regular subunit. In some embodiments, in any of the DP2-DPn fractions, more than 99%, 90%, 80%, 70%, 60%, 50%, 40%, or 30% of the anhydrosubunits are associated with a single regular subunit. In some embodiments, in each of the DP2-DPn fractions, more than 99%, 90%, 80%, 70%, 60%, 50%, 40%, or 30% of the anhydro subunits are associated with a single regular subunit.

In some embodiments, in any fraction of the oligosaccharide preparation that has a degree of polymerization of 2 or more (ie, the DP2-DPn fraction), the anhydro subunit may be located at the end of the oligosaccharide chain. In some embodiments, in any fraction of the oligosaccharide preparation that has a degree of polymerization of 3 or more (ie, the DP3-DPn fraction), the anhydro subunit may be at a position that is not the end of the oligosaccharide chain. In some embodiments, in the DP2-DPn fractions, at least one subunit is located at the end of the oligosaccharide chain. In some embodiments, greater than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 30% anhydrosubunits in the DP2-DPn fractions, they are located at the end of the oligosaccharide chain. In some embodiments, more than 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the anhydrosubunits in the oligosaccharide preparation are located at the end of the oligosaccharide chain. In some embodiments, more than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the anhydrosubunit-containing oligosaccharides include an anhydrosubunit at the end of the chain. In some embodiments, more than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the anhydro subunit-containing oligosaccharides include an anhydro subunit at the end of the chain .

E. Glycosidic bonds

In some embodiments, the oligosaccharide preparation described herein contains multiple glycosidic linkages. The type and distribution of glycosidic bonds may depend on the source and method of production of the oligosaccharide drug. In some embodiments, the type and distribution of various glycosidic linkages can be determined and/or detected by any suitable methods known in the art, such as NMR. For example, in some embodiments, glycosidic linkages are determined and/or detected using ¹ H-NMR, ¹³ C-NMR, 2D NMR such as 2D JRES, HSQC, HMBC, DOSY, COZY, ECOSY, TOCSY, NOESY or ROESY, or any combination of them. In some embodiments, glycosidic linkages are determined and/or detected, at least in part, by ¹ H-NMR. In some embodiments, glycosidic linkages are determined and/or detected, at least in part, by ¹³ C-NMR. In some embodiments, glycosidic linkages are determined and/or detected, at least in part, by 2D 1H,13C-HSQC NMR.

In some embodiments, an oligosaccharide preparation described herein contains one or more α-(1,2) glycosidic linkages, α-(1,3) glycosidic linkages, α-(1,4) glycosidic linkages, α-(1,6 ) glycosidic bonds, β-(1,2) glycosidic bonds, β-(1,3) glycosidic bonds, β-(1,4) glycosidic bonds, β-(1,6) glycosidic bonds, a-(1,1 )-α-glycosidic bonds, α-(1,1)-β-glycosidic bonds, β-(1,1)-β-glycosidic bonds, or any combination thereof.

In some embodiments, the oligosaccharide preparations have a glycosidic linkage type distribution of about 0 to 60 mole%, about 5 to 55 mole%, about 5 to 50 mole%, about 5 to 45 mole%, about 5 up to 40 mol.%, about 5 to 35 mol.%, about 5 to 30 mol.%, about 5 to 25 mol.%, about 10 to 60 mol.%, about 10 to 55 mol.% , about 10 to 50 mol.%, about 10 to 45 mol.%, about 10 to 40 mol.%, about 10 to 35 mol.%, about 15 to 60 mol.%, about 15 to 55 mol.%, about 15 to 50 mol.%, about

- 19 044359 from 15 to 45 mol.%, from about 15 to 40 mol.%, from about 15 to 35 mol.%, from about 20 to 60 mol.%, from about 20 to 55 mol.%, from about 20 up to 50 mol.%, about 20 to 45 mol.%, about 20 to 40 mol.%, about 20 to 35 mol.%, about 25 to 60 mol.%, about 25 to 55 mol.% , about 25 to 50 mol%, about 25 to 45 mol%, about 25 to 40 mol%, or about 25 to 35 mol% α-(1,6) glycosidic linkages.

In some embodiments, the oligosaccharide preparations have a glycosidic linkage type distribution of about 0 to 50 mole%, about 0 to 40 mole%, about 0 to 35 mole%, about 0 to 30 mole%, about 0 up to 25 mol.%, from about 0 to 20 mol.%, from about 5 to 40 mol.%, from about 5 to 35 mol.%, from about 5 to 30 mol.%, from about 5 to 25 mol.%, about 5 to 20 mole%, about 10 to 40 mole%, about 10 to 35 mole%, about 10 to 20 mole% about 15 to 40 mole%, about 15 to 35 mole% .%, about 15 to 30 mol.%, about 15 to 25 mol.%, or about 15 to 20 mol.% α-(1,3) glycosidic linkages.

In some embodiments, the oligosaccharide preparations have a glycosidic linkage type distribution of about 0 to 40 mole%, about 0 to 35 mole%, about 0 to 30 mole%, about 0 to 25 mole%. mol.%, about 0 to 20 mol.%, about 0 to 15 mol.%, about 0 to 10 mol.%, about 2 to 30 mol.%, about 2 to 25 mol.%, about from 2 to 20 mol.%, from about 2 to 15 mol.%, from about 2 to 10 mol.%, from about 3 to 30 mol.%, from about 3 to 25 mol.%, from about 3 to 20 mol.% .%, about 3 to 15 mol.%, about 3 to 10 mol.%, about 5% to 30 mol.%, about 5 to 25 mol.%, about 5 to 20 mol.%, about from 5 to 15 mol.% or about 5 to 10 mol.% α-(1,2) glycosidic linkages.

In some embodiments, the oligosaccharide preparations have a glycosidic linkage type distribution of about 0 to 40 mole%, about 0 to 30 mole%, about 0 to 25 mole%, about 0 to 20 mole%, about 0 up to 15 mol.%, about 0 to 10 mol.%, or about 0 to 5 mol.% α-(1,4) glycosidic linkages. In some embodiments, the oligosaccharide preparations have a glycosidic linkage type distribution of less than 40 mol.%, less than 30 mol.%, less than 20 mol.%, less than 15 mol.%, less than 10 mol.%, less than 9 mol.%, less than 8 mol. .%, less than 7 mol.%, less than 6 mol.%, less than 5 mol.%, less than 4 mol.%, less than 3 mol.% or less than 2 mol.% α-(1,4) glycosidic bonds.

In some embodiments, the oligosaccharide preparations have a glycosidic linkage type distribution of about 0 to 40 mole%, about 0 to 35 mole%, about 0 to 30 mole%, about 0 to 25 mole%, about 0 to 20 mol.%, about 0 to 15 mol.%, about 0 to 10 mol.%, about 2 to 30 mol.%, about 2 to 25 mol.%, about 2 to 20 mol.% , about 2 to 15 mol.%, about 2 to 10 mol.%, about 5 to 30 mol.%, about 5 to 25 mol.%, about 5 to 20 mol.%, about 5 to 15 mol.%, about 5 to 10 mol.%, about 8 to 30 mol.%, about 8 to 25 mol.%, about 8 to 20 mol.%, about 8 to 15 mol.% or about 10 to 15 mol% β-(1,6) glycosidic linkages.

In some embodiments, the oligosaccharide preparations have a glycosidic linkage type distribution of about 0 to 40 mole%, about 0 to 35 mole%, about 0 to 30 mole%, about 0 to 25 mole%, about 0 to 20 mol.%, about 0 to 15 mol.%, about 0 to 10 mol.%, about 2 to 30 mol.%, about 2 to 25 mol.%, about 2 to 20 mol.% , about 2 to 15 mol.%, about 2 to 10 mol.%, about 3 to 30 mol.%, about 3 to 25 mol.%, about 3 to 20 mol.%, about 3 to 15 mol.%, about 3 to 10 mol.%, about 5 to 30 mol.%, about 5 to 25 mol.%, about 5 to 20 mol.%, about 5 to 15 mol.% or about 5 to 10 mol% β-(1,4) glycosidic linkages.

In some embodiments, the oligosaccharide preparations have a glycosidic linkage type distribution of about 0 to 40 mole%, about 0 to 30 mole%, about 0 to 25 mole%, about 0 to 20 mole%, about 0 to 15 mol.%, about 0 to 10 mol.%, about 0 to 5 mol.%, about 1 to 20 mol.%, about 1 to 15 mol.%, about 1 to 10 mol.% , about 1 to 5 mol.%, about 2 to 20 mol.%, about 2 to 15 mol.%, about 2 to 10 mol.%, or about 2 to 5 mol.% β-(1, 2) glycosidic bonds. In some embodiments, the oligosaccharide preparations have a glycosidic linkage type distribution of less than 40 mol.%, less than 30 mol.%, less than 20 mol.%, less than 15 mol.%, less than 10 mol.%, less than 9 mol.%, less than 8 mol. .%, less than 7 mol.%, less than 6 mol.%, less than 5 mol.%, less than 4 mol.%, less than 3 mol.% or less than 2 mol.% β-(1,2) glycosidic bonds.

In some embodiments, the oligosaccharide preparations have a glycosidic linkage type distribution of about 0 to 40 mole%, about 0 to 30 mole%, about 0 to 25 mole%, about 0 to 20 mole%, about 0 to 15 mol.%, about 0 to 10 mol.%, about 0 to 5 mol.%, about 1 to 20 mol.%, about 1 to 15 mol.%, about 1 to 10 mol.% , about 1 to 5 mol.%, about 2 to 20 mol.%, about 2 to 15 mol.%, about 2 to 10 mol.%, or about 2 to 5 mol.% β-(1, 3) glycosidic bonds. In some embodiments, the oligosaccharide preparations have a glycosidic linkage type distribution of less than 40 mol.%, less than 30 mol.%, less than 20 mol.%, less than 15 mol.%, less than 10 mol.%, less than 9 mol.%, less than 8 mol. .%, less than 7 mol.%, less than 6 mol.%, less than 5 mol.%, less than 4 mol.%, less than 3 mol.% or less than 2 mol.% β-(1,3) glycosidic linkages.

- 20 044359

In some embodiments, the oligosaccharide preparations have a glycosidic linkage type distribution that is different from the glycosidic linkage type distribution in non-synthetic oligosaccharide preparations. For example, in some embodiments, the oligosaccharide preparations have a distribution of glycosidic linkage types that differs from that of the base nutritional compositions. In some embodiments, the basic nutritional compositions contain a natural source of carbohydrates, such as starch and plant fiber. Some of the natural carbohydrate sources have a high percentage of α-(1,4), α-(1,6) and/or β-(1,6) glycosidic linkages. Accordingly, in some embodiments, the oligosaccharide preparations have a lower percentage of α-(1,4) glycosidic linkages than the base nutritional composition. In some embodiments, the oligosaccharide preparations have a lower percentage of α-(1,6) glycosidic linkages than the base nutritional composition. In other embodiments, the oligosaccharide preparations have a higher percentage of α-(1,6) glycosidic linkages than the base nutritional composition. In some embodiments, the oligosaccharide preparations have a lower percentage of β-(1,6) glycosidic linkages than the base nutritional composition. In some embodiments, the oligosaccharide preparation contains glycosidic linkages that are difficult to cleave or hydrolyze by enzymes.

Specifically, in some embodiments, α-(1,2), α-(1,3), α-(1,4), α-(1,6), β-(1,2), β-( 1,3), β-(1,4) and/or β-(1,6) glycosidic bonds in the distribution of the glycosidic bond type of the oligosaccharide preparations described in this application by at least 50 mol.%, by at least 40 mol. .%, at least 30 mol.%, at least 20 mol.%, at least 15 mol.%, at least 10 mol.%, at least 5 mol.%, at least at least 2 mol.% or at least 1 mol.% lower than in the main nutritional composition. In some embodiments, α-(1,2), α(1,3), α-(1,4), α-(1,6), β-(1,2), β-(1,3) , β-(1,4) and/or β-(1,6) glycosidic bonds in the distribution of glycosidic bond type of oligosaccharide preparations by at least 50 mol.%, at least 40 mol.%, at least 30 mol.%, at least 20 mol.%, at least 15 mol.%, at least 10 mol.%, at least 5 mol.%, at least 2 mol.% or according at least 1 mol.% higher than in the main nutritional composition.

One skilled in the art will appreciate that certain types of glycosidic linkages may not be applicable to oligosaccharides containing a certain type of monosaccharide. For example, in some embodiments, the oligosaccharide preparation contains α-(1,2) glycosidic linkages and α-(1,6) glycosidic linkages. In other embodiments, the oligosaccharide preparation contains α-(1,2) glycosidic linkages and β-(1,3) glycosidic linkages. In some embodiments, the oligosaccharide preparation contains α-(1,2) glycosidic linkages, α-(1,3) glycosidic linkages, and β-(1,6) glycosidic linkages. In some embodiments, the oligosaccharide preparation contains α-(1,2) glycosidic linkages, α-(1,3) glycosidic linkages, α-(1,4) glycosidic linkages, α-(1,6) glycosidic linkages, β(1 ,2) glycosidic bonds, β-(1,3) glycosidic bonds, β-(1,4) glycosidic bonds, and β-(1,6) glycosidic bonds.

F. Molecular weight

The molecular weight and molecular weight distribution of the oligosaccharide preparations described herein can be determined by any suitable analytical means and instruments, such as the end group method, osmotic pressure (osmometry), ultracentrifugation, viscosity measurement, light scattering method, SEC, SEC-MALLS, PFP , A4F, HPLC and mass spectrometry. In some embodiments, molecular weight and molecular weight distribution are determined by mass spectrometry, such as MALDI-MS, LC-MS, or GC-MS. In some embodiments, molecular weight and molecular weight distribution are determined using size exclusion chromatography (SEC), such as gel permeation chromatography (GPC). In other embodiments, the molecular weight and molecular weight distribution are determined using HPLC. In some embodiments, molecular weight and molecular weight distribution are determined using MALDI-MS.

In some embodiments, the oligosaccharide drug described herein has a weight average molecular weight of about 100 to 10,000 g/mol, about 200 to 8,000 g/mol, about 300 to 5,000 g/mol, about 500 to 5,000 g/mol, about 700 to 5000 g/mol, about 900 to 5000 g/mol, about 1100 to 5000 g/mol, about 1300 to 5000 g/mol, about 1500 to 5000 g/mol, about 1700 to 5000 g/mol, about 300 to 4500 g/mol, about 500 to 4500 g/mol, about 700 to 4500 g/mol, about 900 to 4500 g/mol, about 1100 to 4500 g/mol, about 1300 to 4500 g/mol, about 1500 to 4500 g/mol, about 1700 to 4500 g/mol, about 1900 to 4500 g/mol, about 300 to 4000 g/mol, about 500 to 4000 g /mol, from about 700 to 4000 g/mol, from about 900 to 4000 g/mol, from about 1100 to 4000 g/mol, from about 1300 to 4000 g/mol, from about 1500 to 4000 g/mol, from about 1700 to 4000 g/mol, about 1900 to 4000 g/mol, about 300 to 3000 g/mol, about 500 to 3000 g/mol, about 700 to 3000 g/mol, about 900 to 3000

- 21 044359 g/mol, from about 1100 to 3000 g/mol, from about 1300 to 3000 g/mol, from about 1500 to 3000 g/mol, from about 1700 to 3000 g/mol, from about 1900 to 3000 g/ mole, from about 2100 to 3000 g/mol, from about 300 to 2500 g/mol, from about 500 to 2500 g/mol, from about 700 to 2500 g/mol, from about 900 to 2500 g/mol, from about 1100 up to 2500 g/mol, about 1300 to 2500 g/mol, about 1500 to 2500 g/mol, about 1700 to 2500 g/mol, about 1900 to 2500 g/mol, about 2100 to 2500 g/mol , about 300 to 1500 g/mol, about 500 to 1500 g/mol, about 700 to 1500 g/mol, about 900 to 1500 g/mol, about 1100 to 1500 g/mol, about 1300 to 1500 g /mol, about 2000 to 2800 g/mol, about 2100 to 2700 g/mol, about 2200 to 2600 g/mol, about 2300 to 2500 g/mol, or about 2320 to 2420 g/mol. In some embodiments, the weight average molecular weight of the oligosaccharide drug is about 2000 to 2800 g/mol, about 2100 to 2700 g/mol, about 2200 to 2600 g/mol, about 2300 to 2500 g/mol, or about 2320 up to 2420 g/mol. In some embodiments, the oligosaccharide drug has a weight average molecular weight ranging from at least 500 g/mol, 750 g/mol, 1000 g/mol, or 1500 g/mol to no more than 1750 g/mol, 2000 g/mol, 2250 g /mol, 2500 g/mol or 3000 g/mol. In some embodiments, the weight average molecular weight of an oligosaccharide drug described herein is determined using HPLC according to Example 9.

In some embodiments, the oligosaccharide drug described herein has a number average molecular weight of about 100 to 10,000 g/mol, about 200 to 8,000 g/mol, about 300 to 5,000 g/mol, about 500 to 5,000 g/mol, about 700 to 5000 g/mol, about 900 to 5000 g/mol, about 1100 to 5000 g/mol, about 1300 to 5000 g/mol, about 1500 to 5000 g/mol, about 1700 to 5000 g/mol, about 300 to 4500 g/mol, about 500 to 4500 g/mol, about 700 to 4500 g/mol, about 900 to 4500 g/mol, about 1100 to 4500 g/mol, about from 1300 to 4500 g/mol, from about 1500 to 4500 g/mol, from about 1700 to 4500 g/mol, from about 1900 to 4500 g/mol, from about 300 to 4000 g/mol, from about 500 to 4000 g/ mole, about 700 to 4000 g/mol, about 900 to 4000 g/mol, about 1100 to 4000 g/mol, about 1300 to 4000 g/mol, about 1500 to 4000 g/mol, about 1700 to 4000 g/mol, from about 1900 to 4000 g/mol, from about 300 to 3000 g/mol, from about 500 to 3000 g/mol, from about 700 to 3000 g/mol, from about 900 to 3000 g/mol, from about 1100 to 3000 g/mol, about 1300 to 3000 g/mol, about 1500 to 3000 g/mol, about 1700 to 3000 g/mol, about 1900 to 3000 g/mol, about 2100 to 3000 g/mol, about 300 to 2500 g/mol, about 500 to 2500 g/mol, about 700 to 2500 g/mol, about 900 to 2500 g/mol, about 1100 to 2500 g/mol, about 1300 to 2500 g /mol, from about 1500 to 2500 g/mol, from about 1700 to 2500 g/mol, from about 1900 to 2500 g/mol, from about 2100 to 2500 g/mol, from about 300 to 2000 g/mol, from about 500 to 2000 g/mol, about 700 to 2000 g/mol, about 900 to 2000 g/mol, about 1100 to 2000 g/mol, about 300 to 1500 g/mol, about 500 to 1500 g/mol mole, from about 700 to 1500 g/mol, from about 900 to 1500 g/mol, from about 1100 to 1500 g/mol, from about 1300 to 1500 g/mol, from about 1000 to 2000 g/mol, from about 1100 to 1900 g/mol, about 1200 to 1800 g/mol, about 1300 to 1700 g/mol, about 1400 to 1600 g/mol, or about 1450 to 1550 g/mol. In some embodiments, the number average molecular weight of the oligosaccharide drug is about 1000 to 2000 g/mol, about 1100 to 1900 g/mol, about 1200 to 1800 g/mol, about 1300 to 1700 g/mol, about 1400 to 1600 g/mol or about 1450 to 1550 g/mol. In some embodiments, the oligosaccharide drug has a number average molecular weight ranging from at least 500 g/mol, 750 g/mol, 1000 g/mol, or 1500 g/mol to no more than 1750 g/mol, 2000 g/mol, 2250 g /mol, 2500 g/mol or 3000 g/mol. In some embodiments, the number average molecular weight of an oligosaccharide drug described herein is determined using HPLC according to Example 9.

G. Types of Oligosaccharides

The types of oligosaccharides present in the oligosaccharide preparation may depend on the type of one or more feed sugars. For example, in some embodiments, the oligosaccharide preparations contain a glucooligosaccharide when the feed sugars contain glucose. For example, in some embodiments, the oligosaccharide preparations contain galacto-oligosaccharide when the feed sugars contain galactose. In another example, in some embodiments, the oligosaccharide preparations contain glucogalactooligosaccharides, where feed sugars include galactose and glucose.

In some embodiments, the oligosaccharide preparations described herein contain one or more types of monosaccharide subunits. In some embodiments, the oligosaccharide preparation may contain oligosaccharides with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more different types of monosaccharide subunits.

In some embodiments, the oligosaccharide preparation contains oligosaccharides with 1, 2, 3, or 4 different types of monosaccharide subunits. In some embodiments, the oligo-saccharide preparation includes oligosaccharides with 1, 2, or 3 different types of monosaccharide subunits. In some embodiments, the oligosaccharide preparation contains oligosaccharides with 3 different types of monosaccharide subunits. In some embodiments, the oligosaccharide preparation includes oligosaccharides with 2 different types of monosaccharide subunits. In some embodiments, the oligosaccharide preparation includes one type of monosaccharide subunit.

In some embodiments, the oligosaccharide preparation includes various types of oligosaccharides, with each oligosaccharide molecule independently comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different types of monosaccharide subunits. In some embodiments, the oligosaccharide preparation described herein contains 10 ² , 103, 104, 105 or more different types of oligosaccharides. In some embodiments, some of the oligosaccharides in the preparation contain one type of monosaccharide subunit, and some of the other oligosaccharides in the same preparation contain two or more types of monosaccharide subunits. For example, in some embodiments, when the feed sugars are glucose and galactose, the oligosaccharide preparation may include oligosaccharides that contain only glucose subunits, oligosaccharides that contain only galactose subunits, oligosaccharides that contain glucose and galactose subunits in varying ratios, or any a combination of them.

In some embodiments, any or all n fractions of the oligosaccharide preparation contain different types of oligosaccharide subunits, where each oligosaccharide independently includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different types of monosaccharide subunits. In some embodiments, some of the oligosaccharides in the drug fraction contain one type of monosaccharide subunit, and some of the other oligosaccharides in the same drug fraction contain two or more types of monosaccharide subunits.

In some embodiments, an oligosaccharide preparation described herein contains one or more monosaccharide subunits selected from the group consisting of triose, tetrose, pentose, hexose, heptose, and any combination thereof, wherein each is triose, tetrose, pentose, hexose, or heptose subunits independently and, if necessary, functionalized and/or replaced by one of its corresponding anhydrosubunits. In some embodiments, the corresponding anhydrous subunit is the product of reversible thermal dehydration of a monosaccharide subunit. In some embodiments, the corresponding anhydro subunit is a caramelization product of a monosaccharide subunit.

In some embodiments, an oligosaccharide preparation described herein comprises pentose subunits, hexose subunits, or any combination thereof, wherein each of the pentose or hexose subunits is independently and optionally functionalized and/or replaced by one of its corresponding anhydro subunits. In some embodiments, the oligosaccharide preparation contains hexose subunits, wherein each of said hexose subunits is independently and optionally replaced by one of its corresponding anhydrosubunits.

In the context of the present invention, tetrose refers to a monosaccharide with four carbon atoms, such as erythrose, threose and erythrulose. In the context of the present invention, pentose refers to a monosaccharide with five carbon atoms, such as arabinose, lyxose, ribose and xylose. In the context of the present invention, hexose refers to a monosaccharide with six carbon atoms, such as allose, altrose, glucose, mannose, gulose, idose, galactose, talose, psicose, fructose, sorbose and tagatose. In the context of the present invention, heptose refers to a monosaccharide with seven carbon atoms, such as sedoheptulose and mannoheptulose.

In some embodiments, an oligosaccharide preparation described herein comprises a glucose subunit, wherein at least one glucose subunit is optionally replaced by an anhydroglucose subunit. In some embodiments, an oligosaccharide preparation described herein comprises a galactose subunit, wherein at least one galactose subunit is optionally replaced by an anhydrogalactose subunit. In some embodiments, the oligosaccharide preparation described herein contains galactose and glucose subunits, wherein at least one galactose subunit or at least one glucose subunit is optionally replaced by one of its corresponding anhydro subunits. In some embodiments, an oligosaccharide preparation described herein comprises fructose and glucose subunits, wherein at least one fructose subunit or at least one glucose subunit is optionally replaced by one of its corresponding anhydrous subunits. In some embodiments, the oligosaccharide preparation described herein comprises mannose and glucose subunits, wherein at least one mannose subunit or at least one glucose subunit is optionally replaced by one of its corresponding anhydrosubunits.

In some embodiments, an oligosaccharide drug described herein includes a glucogalactose-oligosaccharide drug, a gluco-oligosaccharide drug, a galacto-oligo

- 23 044359 saccharide drug, fructooligosaccharide drug, mannooligosaccharide drug, arabinooligosaccharide drug, xylooligosaccharide drug, glucofructooligosaccharide drug, glucomannooligosaccharide drug, glucoarabinooligosaccharide drug, glucoxylooligosaccharide drug, galactofructooligosaccharide ny drug, mannooligosaccharide drug, galactoarabinooligosaccharide drug, galactoxylooligosaccharide drug, fructomannooligosaccharide drug, fructoorabinooligosaccharide drug, fructoxylooligosaccharide drug . , glucogalactofructomannoarabinoxylooligosaccharide drug, or any combination thereof; where each of the monosaccharide subunits in the composition of the drug is independently and, if necessary, functionalized and/or replaced by one of its corresponding anhydrous subunits.

In some embodiments, the oligosaccharide preparation described herein contains more than 99% glucose subunits by weight. In some embodiments, the oligosaccharide preparation contains only glucose subunits.

In some embodiments, the oligosaccharide preparation described herein contains about 45% to 55% glucose subunits and about 55% to 45% galactose subunits by weight. In some embodiments, the oligosaccharide preparation contains about 50% glucose subunits and 50% galactose subunits by weight.

In some embodiments, the oligosaccharide preparation described herein contains about 80 to 95% glucose subunits and about 20 to 5% mannose subunits by weight. In some embodiments, the oligosaccharide preparation contains about 85 to 90% glucose subunits and about 15 to 10% mannose subunits by weight.

In some embodiments, the oligosaccharide preparation described herein contains about 80 to 95% glucose subunits and about 20 to 5% galactose subunits by weight. In some embodiments, the oligosaccharide preparation contains about 85 to 90% glucose subunits and about 15 to 10% galactose subunits by weight.

In some embodiments, the oligosaccharide preparation described herein contains from about 80 to 95% glucose subunits, from 0 to 8% galactose subunits, and from 5 to 20% mannose subunits by weight. In some embodiments, the oligosaccharide preparation contains about 80 to 90% glucose subunits, 1 to 5% galactose subunits, and 10 to 15% mannose subunits by weight.

In some embodiments, the oligosaccharide preparation described herein contains from about 1 to 100 wt%, about 50 to 100 wt%, about 80 to 98 wt%, or about 85 to 95 wt% glucose subunits , or any range in between. In some embodiments, the galactose subunits are present in the oligosaccharide preparation described herein in an amount of about 0 to 90 wt%, about 1 to 50 wt%, about 2 to 20 wt%, or about 5 to 15 wt.%, or any range in between. In some embodiments, mannose subunits are present in the oligosaccharide preparation described herein in an amount of about 0 to 90 wt%, about 1 to 50 wt%, about 2 to 20 wt%, or about 5 to 15 wt.%, or any range in between.

In some embodiments, the oligosaccharide preparation described herein has a monosaccharide subunit composition as shown in Table. 28.

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Table 28. Approximate composition of oligosaccharide preparations

No. oligosaccharides of the composition Glucose and anhydroglucose subunits (wt.%) Galactose and anhydrogalactose subunits (wt.%) Mannose and anhydromannose subunits (wt.%) Fructose and anhydrofructose subunits (wt.%) 1 87.5 12.5 0 0 2 100 0 0 0 3 85 2.5 12.5 0 4 87.5 0 12.5 0 5 50 50 0 0 6 75 0 25 0 7 9 6 0 0 8 90 0 10 0 9 95 5 0 0 10 97.5 2.5 0 0 eleven 85 5 10 0 12 85 1.5 13.5 0 13 80 10 10 0 14 85 0 15 0 15 85 15 0 0 16 87.5 0 0 12.5

H. D-shape and L-shape

In some embodiments, at least one monosaccharide subunit in the oligosaccharide is in the L form. In some embodiments, at least one monosaccharide subunit in the oligosaccharide is in the D form. In some embodiments, the monosaccharide subunits in an oligosaccharide preparation described herein are in their naturally occurring abundant form, such as D-glucose, D-xylose, and L-arabinose.

In some embodiments, an oligosaccharide preparation described herein includes a mixture of L and D forms of monosaccharide subunits. In some embodiments, the ratio of monosaccharide subunits L- to D- or D- to L- form is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, approximately 1:7, approximately 1:8, approximately 1:9, approximately 1:10, approximately 1:12, approximately 1:14, approximately 1:16, approximately 1:18, approximately 1:20, approximately 1:25, approximately 1:30, approximately 1:35, approximately 1:40, approximately 1:45, approximately 1:50, approximately 1:55, approximately 1:60, approximately 1:65, approximately 1:70, approximately 1:75, about 1:80, about 1:85, about 1:90, about 1:100 or about 1:150.

I. Functionalized oligosaccharides

In some embodiments, one or more oligosaccharides in an oligosaccharide preparation described herein are independently functionalized. Functionalized oligosaccharides can be prepared, for example, by combining one or more sugars with one or more functionalizing compounds in the presence of a catalyst. Methods for preparing functionalized oligosaccharides are described in WO 2012/118767, WO 2014/031956 and WO/2016/122887, which are incorporated herein by reference in their entirety and for their disclosure.

In some embodiments, the functionalizing compound contains one or more acidic groups (eg, -COOH), hydroxyl groups, or N-containing groups (eg, -CN, -NO2, and -N(Ra) ₂ , where Ra represents hydrogen, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, cycloalkyl, aryl, heterocycloalkyl or heteroaryl group), S-containing groups (for example, thiol and sulfate), halides (for example, -Cl), P-containing groups (for example, phosphate) or any of these combination. In some embodiments, the functionalizing compound is linked to at least one monosaccharide subunit via an ether, ester, oxygen-sulfur, amine, or oxygen-phosphorus bond. In some embodiments, one or more functionalizing compounds are linked to the monosaccharide subunit through a single bond. In some embodiments, the at least one functionalizing compound is linked to one or two oligosaccharides by two or more linkages.

It should be understood that for each oligosaccharide in the oligosaccharide preparation, each of the described embodiments is independent and can be combined as if each combination were listed separately; thus, any combination of embodiments is covered by the present disclosure. For example, various embodiments may be grouped into several categories, which include (i) the presence or absence of an anhydrosubunit; (ii) the amount and level of anhydrosubunit; (iii) type of anhydrosubunit species; (iv) location of the anhydro subunit; (v) degree of polymerization; (vi) molecular weight; (vii) the presence or absence of any functional groups; (viii) type of oligosaccharide; (ix) type of glycosidic bond and (x) L- or D

- 25 044359 form; but are not limited to them. Accordingly, the described oligosaccharide preparation includes a variety of oligosaccharides of different types. In some embodiments, an oligosaccharide preparation described herein contains at least 10, ¹⁰² , ¹⁰³ , ¹⁰⁴ , ¹⁰⁵ , ¹⁰⁶ , ¹⁰⁷ , ¹⁰⁸ , ¹⁰⁹ , or ¹⁰¹⁰ different types of oligosaccharides. In some embodiments, the formulation contains at least 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ or 10 ⁹ different types of oligosaccharides. In some embodiments, the formulation contains at least 10 ³ different types of oligosaccharides.

III. Methods for the production of oligosaccharide preparations

In one aspect, this application provides methods for the production of oligosaccharide preparations. In some embodiments, this application provides methods for producing oligosaccharide preparations suitable for use in a nutritional composition, such as an animal feed composition, or for feeding directly to an animal. In one aspect, this application provides methods for producing an oligosaccharide preparation comprising heating an aqueous composition containing one or more feed sugars and a catalyst to a temperature and for a time sufficient to induce polymerization, wherein the catalyst is selected from the group consisting of: (+) -camphor-10-sulfonic acid; 2-pyridine sulfonic acid; 3pyridine sulfonic acid; 8-hydroxy-5-quinoline sulfonic acid hydrate; a-hydroxy-2pyridinemethanesulfonic acid; (c)-camphor-10-sulfonic acid; butylphosphonic acid; diphenylphosphinic acid; hexylphosphonic acid; methylphosphonic acid; phenylphosphinic acid; phenylphosphonic acid; tert-butylphosphonic acid; (S)-VAPOL-hydrogen phosphate; 6quinoline sulfonic acid, 3-(1-pyridino)-1-propanesulfonate; 2-(2-pyridinyl)ethanesulfonic acid; 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p'-disulfonic acid monosodium salt hydrate; 1,1'-binaphthyl-2,2'-diylhydrogenphosphate; bis(4-methoxyphenyl)phosphinic acid; phenyl(3,5xylyl)phosphinic acid; L-cysteine acid monohydrate; poly(styrenesulfonic acid co-divinylbenzene); lysine; ethanesulfonic acid; ethanesulfonic acid; isethionic acid; homocysteine acid; HEPBS (K-(2-hydroxyethyl)piperazine-N-(4-butanesulfonic acid)); HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid); 2-hydroxy-3morpholinopropanesulfonic acid; 2-(K-morpholino)ethanesulfonic acid; methanesulfonic acid; metaniazide; naphthalene-1-sulfonic acid; naphthalene-2-sulfonic acid; perfluorobutanesulfonic acid; 6-sulfoquinovose; triflic acid; 2-aminoethanesulfonic acid; benzoic acid; chloroacetic acid; trifluoroacetic acid; caproic acid; enanthic acid; caprylic acid; pelargonic acid; lauric acid; palmitic acid; stearic acid; arachidic acid; aspartic acid; glutamic acid; serine; threonine; glutamine; cysteine; glycine; proline; alanine; valina; isoleucine; leucine; methionine; phenylalanine; tyrosine; tryptophan, and wherein the oligosaccharide preparation contains at least n oligosaccharide fractions, each of which has a different degree of polymerization selected from 1 (fraction DP1) to n (fraction DPn), where n is an integer equal to 2 or more.

In some embodiments, n is an integer equal to 3 or more. In some embodiments, n is an integer ranging from 1 to 100, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or 50. In some embodiments, polymerization of feed sugars is achieved by step polymerization. In some embodiments, polymerization of feed sugars is achieved by polycondensation.

A. Feed sugar

In some embodiments, the method for producing oligosaccharide preparations described herein includes heating one or more types of feed sugars. In some embodiments, the one or more feed sugars comprise monosaccharides, disaccharides, trisaccharides, tetrasaccharides, or any mixtures thereof.

In some embodiments, the one or more feed sugars comprise glucose. In some embodiments, the one or more feed sugars comprise glucose and galactose. In some embodiments, the one or more feed sugars comprise glucose, xylose, and galactose. In some embodiments, the one or more feed sugars comprise glucose and mannose. In some embodiments, the one or more feed sugars comprise glucose and fructose. In some embodiments, the one or more feed sugars comprise glucose, fructose, and galactose. In some embodiments, the one or more feed sugars comprise glucose, galactose, and mannose.

In some embodiments, the one or more feed sugars comprise disaccharides such as lactose, sucrose, and cellobiose. In some embodiments, the one or more feed sugars comprise trisaccharides such as maltotriose or raffinose. In some embodiments, the one or more feed sugars comprise glucose, mannose, galactose, xylose, maltodextrin, arabinose, or galactose, or any combination thereof. In some embodiments, the one or more feed sugars comprise a sugar syrup such as corn syrup. IN

- 26 044359 In some embodiments, one or more feed sugars comprise glucose and lactose.

In some embodiments, the one or more feed sugars comprise glucose and sucrose.

In some embodiments, the type of feed sugars may influence the resulting oligosaccharide preparations. For example, in some embodiments, when one or more feed sugars are glucose, the resulting oligosaccharide preparations include glucooligosaccharide preparations. In other embodiments, when one or more feed sugars are mannose, the resulting oligosaccharide preparations include manno-oligosaccharide preparations. In some embodiments, where one or more feed sugars comprise glucose and galactose, the resulting oligosaccharide preparations include glucogalacto-oligosaccharide preparations. In yet other embodiments, where one or more feed sugars include xylose, glucose and galactose, the resulting oligosaccharide preparations include glucogalactoxylooligosaccharide preparations.

In some embodiments, each of the one or more feed sugars may independently be in its dehydrated or hydrated form. In some embodiments, the one or more feed sugars comprise glucose, galactose, fructose, mannose, or any combination thereof, and wherein each of glucose, galactose, fructose, or mannose is independently in its monohydrate or dehydrated form. In some embodiments, the one or more feed sugars comprise a monosaccharide monohydrate, such as glucose monohydrate. In some embodiments, the one or more feed sugars comprise a saccharide dihydrate, such as trehalose dihydrate. In some embodiments, the one or more feed sugars comprise at least one sugar in its dehydrated form and at least one sugar in its hydrated form.

In some embodiments, one or more feed sugars may be provided as a sugar solution where the sugars are combined with water and loaded into a reactor. In some embodiments, the sugars may be supplied to the reactor in solid form and combined with water in the reactor. In some embodiments, one or more feed sugars are combined and mixed before water is added. In some embodiments, one or more feed sugars are combined in water and then mixed.

In some embodiments, the method includes combining two or more feed sugars with a catalyst to produce an oligosaccharide preparation. In some embodiments, the two or more feed sugars include glucose, galactose, fructose, mannose, lactose, or any combination thereof. In some embodiments, the method includes combining a mixture of sugars (eg, monosaccharides, disaccharides, and/or trisaccharides) with a catalyst to produce an oligosaccharide preparation. In other embodiments, the method includes combining a mixture of sugars and sugar alcohols with a catalyst to produce an oligosaccharide preparation.

In some embodiments, the one or more feed sugars comprise functionalized or modified sugars. Functionalized or modified sugars may include amino sugars, sugar acids, sugar alcohols, sugar amides, sugar ethers, or any combination thereof. In some embodiments, amino sugars refer to sugar molecules in which the hydroxyl group is replaced by an amino group. Examples of amino sugars include N-acetyl-D-glucosamine, mannosamine, neuraminic acid, muramic acid, N-acetylneuraminic acid, N-acetylmuramic acid, N-acetylgalactosamine, N-acetylmannose, N-glycolylneuraminic acid, acarviosine, D-glucosamine and D-galactosamine. but are not limited to them.

In some embodiments, sugar acids refer to sugars with a carboxyl group. Examples of sugar acids include aldonic acids (such as glyceric acid, xylonic acid, gluconic acid and ascorbic acid), ulosonic acids (such as neuraminic acid and ketodeoxyoctulosonic acid), uronic acids (such as glucuronic acid, galacturonic acid and iduronic acid) and aldaric acids. acids (such as, but not limited to, tartaric acid, mucinic acid and sugar acid).

In some embodiments, sugar alcohols refer to sugar-derived polyols. Examples of sugar alcohols include, but are not limited to, ethylene glycol, arabitol, glycerin, erythritol, threitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, and volemitol.

In some embodiments, sugar amides refer to sugar molecules that contain a -C(=O)-N- group. In some embodiments, sugar ethers refer to sugar molecules that contain an ester bond, such as glucosides.

In some embodiments, functionalized or modified sugar acids include glucosamine, N-acetylglucosamine, glucuronic acid, galacturonic acid, glucitol, xylitol, mannitol, sorbitol. In some embodiments, the one or more feed sugars include deoxysugars such as fucose, rhamnose, deoxyribose, or fuculose.

- 27 044359

In some embodiments, the method of producing an oligosaccharide preparation described herein is carried out on a gram scale. In some embodiments, the method of producing an oligosaccharide preparation described herein is carried out on a kilogram or larger scale. Accordingly, in some embodiments, the method includes heating an aqueous composition containing one or more feed sugars in an amount greater than 0.5, greater than 1, greater than 2, greater than 3, greater than 4, greater than 5, greater than 6, greater than 7, greater than 9, more 10, more than 100 or more than 1000 kg. In some embodiments, the method includes heating an aqueous composition containing one or more feed sugars in an amount of not more than 0.5, 1, 2, 3, 4, 5, 6, 7, 9, 10, 100, 1000 or 1500 kg. In some embodiments, the method includes heating an aqueous composition containing one or more feed sugars in an amount greater than 1 kg.

B. Catalysts

In some embodiments, the catalyst provided herein includes one or more acids. In some embodiments, the catalyst provided herein includes a mineral acid, a carboxylic acid; amino acid; sulfonic acid; boronic acid; phosphonic acid; phosphinic acid; sulfuric acid; phosphoric acid; poly(styrenesulfonic acid-co-vinylbenzyl-imidazolium sulfate-co-divinylbenzene); poly(styrenesulfonic acid-co-divinylbenzene); (+)-camphor-10-sulfonic acid; 2pyridine sulfonic acid; 3-pyridine sulfonic acid; 8-hydroxy-5-quinoline sulfonic acid hydrate; a-hydroxy-2-pyridinemethanesulfonic acid; (c)-camphor-10-sulfonic acid; butylphosphonic acid; diphenylphosphinic acid; hexylphosphonic acid; methylphosphonic acid; phenylphosphinic acid; phenylphosphonic acid; tert-butylphosphonic acid; (S)-VAPOL-hydrogen phosphate; 6-quinoline sulfonic acid; 3-(1-pyridino)-1-propanesulfonate; 2-(2pyridinyl)ethanesulfonic acid; 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p'-disulfonic acid monosodium salt hydrate; 1,1'-binaphthyl-2,2'-diylhydrogen phosphate; bis(4-methoxyphenyl)phosphinic acid; phenyl(3,5-xylyl)phosphinic acid; L-cysteine acid monohydrate; acetic acid; propionic acid; butanoic acid; glutamic acid; lysine, ethanedisulfonic acid; ethanesulfonic acid; isethionic acid; homocysteine acid; HEPBS N-(2-hydroxyethyl)piperazine-N-(4-butanesulfonic acid)); HEPES (4-(2-hydroxyethyl)-1piperazineethanesulfonic acid); 2-hydroxy-3-morpholinopropanesulfonic acid; 2-(Nmorpholino)ethanesulfonic acid; methanesulfonic acid; metaniazide; naphthalene-1sulfonic acid; naphthalene-2-sulfonic acid; perfluorobutanesulfonic acid; 6sulfoquinovose; triflic acid; 2-aminoethanesulfonic acid; benzoic acid; chloroacetic acid; trifluoroacetic acid; caproic acid; enanthic acid; caprylic acid; pelargonic acid; lauric acid; palmitic acid; stearic acid; arachidic acid; aspartic acid; glutamic acid; serine; threonine; glutamine; cysteine; glycine; proline; alanine; valine; isoleucine; leucine; methionine; phenylalanine; tyrosine; tryptophan; polymeric acid; carbon-based acid; or any combination thereof.

In some embodiments, the catalyst provided herein includes: (+)-camphor-10-sulfonic acid; 2-pyridine sulfonic acid; 3-pyridine sulfonic acid; 8-hydroxy-5-quinoline sulfonic acid hydrate; a-hydroxy-2-pyridinemethanesulfonic acid; (c)-camphor-10-sulfonic acid; butylphosphonic acid; diphenylphosphinic acid; hexylphosphonic acid; methylphosphonic acid; phenylphosphinic acid; phenylphosphonic acid; tert-butylphosphonic acid; (S)-VAPOL-hydrogen phosphate; 6-quinoline sulfonic acid, 3(1-pyridino)-1-propanesulfonate; 2-(2-pyridinyl)ethanesulfonic acid; 3-(2-pyridyl)-5,6diphenyl-1,2,4-triazine-p,p'-disulfonic acid monosodium salt hydrate; 1,1'-binaphthyl-2,2'diylhydrogen phosphate; bis(4-methoxyphenyl)phosphinic acid; phenyl(3,5-xylyl)phosphinic acid; L-cysteine acid monohydrate; poly(styrenesulfonic acid-co-divinylbenzene); lysine; ethanesulfonic acid; ethanesulfonic acid; isethionic acid; homocysteine acid; HEPBS (N-(2-hydroxyethyl)piperazine-N'-(4-butanesulfonic acid)); HEPES (4-(2-hydroxyethyl)-1piperazineethanesulfonic acid); 2-hydroxy-3-morpholinopropanesulfonic acid; 2-(Nmorpholino)ethanesulfonic acid; methanesulfonic acid; metaniazide; naphthalene-1sulfonic acid; naphthalene-2-sulfonic acid; perfluorobutanesulfonic acid; 6sulfoquinovose; triflic acid; 2-aminoethanesulfonic acid; benzoic acid; chloroacetic acid; trifluoroacetic acid; caproic acid; enanthic acid; caprylic acid; pelargonic acid; lauric acid; palmitic acid; stearic acid; arachidic acid; aspartic acid; glutamic acid; serine; threonine; glutamine; cysteine; glycine; proline; alanine; valine; isoleucine; leucine; methionine; phenylalanine; tyrosine; tryptophan; or any combination thereof.

In some embodiments, the catalyst provided herein is (+)-camphor-10-sulfonic acid. In some embodiments, the catalyst provided herein is 2-pyridine sulfonic acid. In some

- 28 044359 embodiments, the catalyst proposed in this application is 3-pyridine sulfonic acid. In some embodiments, the catalyst provided herein is 8-hydroxy-5-quinoline sulfonic acid hydrate. In some embodiments, the catalyst provided herein is αhydroxy-2-pyridinemethanesulfonic acid. In some embodiments, the catalyst provided herein is (c)-camphor-10-sulfonic acid. In some embodiments, the catalyst provided herein is butylphosphonic acid. In some embodiments, the catalyst provided herein is diphenylphosphinic acid. In some embodiments, the catalyst provided herein is a hexylphosphonic acid. In some embodiments, the catalyst provided herein is methylphosphonic acid. In some embodiments, the catalyst provided herein is phenylphosphinic acid. In some embodiments, the catalyst provided herein is a phenylphosphonic acid. In some embodiments, the catalyst provided herein is tert-butylphosphonic acid. In some embodiments, the catalyst provided herein is (S)-VAPOL hydrogen phosphate. In some embodiments, the catalyst provided herein is 6-quinoline sulfonic acid. In some embodiments, the catalyst provided herein is 3-(1-pyridino)-1-propanesulfonate. In some embodiments, the catalyst provided herein is 2-(2-pyridinyl)ethanesulfonic acid. In some embodiments, the catalyst provided herein is 3-(2pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p'-disulfonic acid monosodium salt hydrate. In some embodiments, the catalyst provided herein is 1'binaphthyl-2,2'-diyl hydrogen phosphate. In some embodiments, the catalyst provided herein is bis(4-methoxyphenyl)phosphinic acid. In some embodiments, the catalyst provided herein is phenyl(3,5xylyl)phosphinic acid. In some embodiments, the catalyst provided herein is L-cysteine acid monohydrate. In some embodiments, the catalyst provided herein is poly(styrenesulfonic acid-co-divinylbenzene). In some embodiments, the catalyst provided herein is lysine.

In some embodiments, the catalyst is ethanedisulfonic acid. In some embodiments, the catalyst is ethanesulfonic acid. In some embodiments, the catalyst is isethionic acid. In some embodiments, the catalyst is homocysteine acid. In some embodiments, the catalyst is HEPBS (K-(2-hydroxyethyl)piperazine-N-(4butanesulfonic acid)). In some embodiments, the catalyst is HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). In some embodiments, the catalyst is 2-hydroxy-3-morpholinopropanesulfonic acid. In some embodiments, the catalyst is 2-(H-morpholino)ethanesulfonic acid. In some embodiments, the catalyst is methanesulfonic acid. In embodiments, the catalyst is naphthalene-1-sulfonic acid. In some embodiments, the catalyst is a metathaniazide. In some embodiments, the catalyst is naphthalene-2-sulfonic acid. In some embodiments, the catalyst is perfluorobutanesulfonic acid. In some embodiments, the catalyst is 6-sulfoquinovose. In some embodiments, the catalyst is triflic acid. In some embodiments, the catalyst is 2-aminoethanesulfonic acid. In some embodiments, the catalyst is benzoic acid. In some embodiments, the catalyst is chloroacetic acid. In some embodiments, the catalyst is trifluoroacetic acid. In some embodiments, the catalyst is caproic acid. In some embodiments, the catalyst is an enanthic acid. In some embodiments, the catalyst is caprylic acid. In some embodiments, the catalyst is pelargonic acid. In some embodiments, the catalyst is lauric acid. In some embodiments, the catalyst is palmitic acid. In some embodiments, the catalyst is stearic acid. In some embodiments, the catalyst is arachidic acid. In some embodiments, the catalyst is aspartic acid. In some embodiments, the catalyst is glutamic acid. In some embodiments it is possible to

- 29 044359 The catalyst is serine. In some embodiments, the catalyst is threonine. In some embodiments, the catalyst is glutamine. In some embodiments, the catalyst is cysteine. In some embodiments, the catalyst is glycine. In some embodiments, the catalyst is proline. In some embodiments, the catalyst is alanine. In some embodiments, the catalyst is valine. In some embodiments, the catalyst is isoleucine. In some embodiments, the catalyst is leucine. In some embodiments, the catalyst is methionine. In some embodiments, the catalyst is phenylalanine. In some embodiments, the catalyst is tyrosine. In some embodiments, the catalyst is tryptophan.

In some embodiments, the catalyst provided herein is a polymer or carbon-supported catalyst disclosed in WO 2016122887, which is incorporated herein by reference in its entirety and for its disclosure.

In some embodiments, the catalyst provided herein is present in an amount of about 0.01 to 5%, about 0.02 to 4%, about 0.03 to 3%, or about 0.05 to 2% of one or several feed sugars by dry matter weight. In some embodiments, the catalyst provided herein is present in an amount of about 1 to 2% of the one or more feed sugars by dry weight. In some embodiments, the catalyst provided herein is present in an amount of about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1 .1%, approximately 1.2%, approximately 1.3%, approximately 1.4%, approximately 1.5%, approximately 1.6%, approximately 1.7%, approximately 1.8%, approximately 1.9 %, approximately 2.0%, approximately 2.1%, approximately 2.2%, approximately 2.3%, approximately 2.4%, approximately 2.5%, approximately 2.6%, approximately 2.7%, about 2.8%, about 2.9%, or about 3.0% of one or more feed sugars by dry matter weight.

In some embodiments, the catalyst provided herein is present in an amount of about 0.01 to 5%, about 0.02 to 4%, about 0.03 to 3%, or about 0.05 to 2% of the aqueous composition by weight of dry matter. In some embodiments, the catalyst provided herein is present in an amount of about 1 to 2% of the aqueous composition by dry weight. In some embodiments, the catalyst provided herein is present in an amount of about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4 %, approximately 1.5%, approximately 1.6%, approximately 1.7%, approximately 1.8%, approximately 1.9%, approximately 2.0%, approximately 2.1%, approximately 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, or about 3.0% of the aqueous composition by weight of dry matter.

In some embodiments, the catalyst provided herein is a combination of two or more different catalysts. In some embodiments, the catalyst includes a recyclable catalyst, such as resins and polymer catalysts, and a non-recyclable catalyst. In some embodiments, when the catalyst includes at least two different catalysts, each of the catalysts is present in the amount provided herein. In other embodiments, where the catalyst includes at least two different catalysts, the at least two different catalysts are present together in the amount specified herein.

In some embodiments, the catalyst is added to the aqueous composition in dry form. In other embodiments, the catalyst is added to the aqueous composition in wet form, for example, in an aqueous solution. In some embodiments, the catalyst is combined with one or more feed sugars before adding water. In other embodiments, the catalyst is dissolved in water before it is combined with one or more feed sugars. In some embodiments, the method provided herein includes preparing an aqueous composition by combining one or more feed sugars in dehydrated form and a catalyst in wet form (eg, as an aqueous solution).

C. Adding water

In some embodiments, a method for producing oligosaccharide preparations described herein includes adding water to form an aqueous composition. In some embodiments, all or a portion of the water in the aqueous composition is added as free water. In other embodiments, all of the water in the aqueous composition is added as bound water, such as saccharide mono- or dihydrate. In some embodiments, all of the water in the aqueous composition is added as bound water in a monosaccharide monohydrate, such as glucose monohydrate. In some embodiments, all or a portion of the water in the aqueous composition is added with the catalyst, that is, through a catalyst solution.

D. Water content

As the methods for producing oligosaccharide preparations progress, water can be produced through the reaction. For example, in some embodiments, water is produced (i) by forming a glycosidic bond, (ii) by forming an anhydro subunit, or (iii) through other mechanisms or sources. Because both sugar condensation and dehydration reactions involve water, in some embodiments, water content affects the composition of the oligosaccharide preparation.

Additionally, in some embodiments, water content affects the viscosity of the aqueous composition, which in turn may affect the mixing efficiency of the aqueous composition. For example, in some embodiments, an excessively viscous aqueous composition may result in undesirable heterogeneous distribution of the catalyst in the aqueous composition. Moreover, in some embodiments, very low water content may cause the aqueous composition to harden, preventing effective mixing. On the other hand, in some other embodiments, extremely high water content may inhibit the sugar condensation reaction and lower the level of anhydrous subunits. Accordingly, the present invention describes a suitable water content for the production of oligosaccharide preparations.

In some embodiments, a method for producing an oligosaccharide preparation described herein includes forming and/or heating an aqueous composition. In some embodiments, the aqueous composition contains from about 0 to 80%, about 0 to 70%, about 0 to 60%, about 0 to 50%, about 0 to 40%, about 0 to 35%, about from 0 to 30%, from about 0 to 25%, from about 0 to 20%, from about 0 to 19%, from about 0 to 18%, from about 0 to 17%, from about 0 to 16%, from about 0 to 15%, about 0 to 14%, about 0 to 13%, about 0 to 12%, about 0 to 11%, about 0 to 10%, about 0 to 9%, about 0 to 8%, about 0 to 7%, about 0 to 6%, about 0 to 5%, about 0 to 4%, about 0 to 3%, about 0 to 2%, or about 0 to 2% 1% water of the total mass. In some embodiments, the aqueous composition contains from about 1 to 20%, about 1 to 18%, about 1 to 16%, about 1 to 14%, about 1 to 12%, about 1 to 10%, about 1 to 8%, about 1 to 6%, or about 1 to 4% water by weight. In some embodiments, the aqueous composition contains about 3 to 16%, about 3 to 14%, about 3 to 12%, about 3 to 10%, about 3 to 8%, about 3 to 6%, about from 5 to 16%, from about 5 to 14%, from about 5 to 12%, from about 5 to 10%, from about 7 to 16%, from about 7 to 14%, from about 7 to 12%, from about 7 to 10% or about 8 to 10% water by weight. In some embodiments, the aqueous composition contains about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11% , about 12%, about 13%, about 14%, or about 15% water by weight. In some embodiments, the aqueous composition contains about 9% water by weight. However, it should be understood that the amount of water in the aqueous composition can be adjusted depending on the reaction conditions and the particular catalyst used. In some embodiments, the water content of the aqueous composition, as described above, is measured at the beginning of the reaction, for example, before heating the feed sugars. In some embodiments, the water content of the aqueous composition described above is measured at the end of the polymerization or condensation reaction. In some embodiments, the water content of the aqueous composition described above is measured as the average water content at the beginning of the reaction and at the end of the reaction.

In some embodiments, the method described herein may further include monitoring the content of water present in the aqueous composition and/or the ratio of water to sugars or catalyst over a period of time. In some embodiments, the method further includes removing at least a portion of the water from the aqueous composition, such as by distillation. Any method known in the art can be used to remove water from the aqueous composition, including, for example, vacuum filtration, vacuum distillation, heat, steam, hot air and/or evaporation.

In some embodiments, the oligosaccharide preparations described herein are hygroscopic. Thus, in some embodiments, the hygroscopicity of feed sugars and oligosaccharides formed during polymerization may affect the rate at which water can be removed from the aqueous composition.

In some embodiments, the method described herein includes removing at least a portion of water from the aqueous composition such that the water content of the aqueous composition is from about 1 to 20%, about 1 to 18%, about 1 to 16%, about 1 to 14%, about 1 to 12%, about 1 to 10%, about 1 to 8%, about 2 to 16%, about 2 to 14%, about 2 to 12%, about 2 to 10%, about 2 to 8%, about 2 to 6%, about 4 to 16%, about 4 to 14%, about 4 to 12%, about 4 to 10%, about 4 to 8%, about 6 to 16%, about 6 to 12%, about 6 to 10%, or about 6 to 8% by weight. In some embodiments, the method includes removing at least a portion of the water from the aqueous composition such that the water content of the aqueous composition is about 2 to 10%, about 2 to 8%, or about 4 to 8% by weight. In some embodiments, the method includes removing at least a portion of water from the aqueous composition

- 31 044359 such that the water content of the aqueous composition is about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9% or about 10% of the total masses. In some embodiments, the method includes removing at least a portion of the water from the aqueous composition such that the water content of the aqueous composition is from about 4% to 8% by weight. In some embodiments, the method includes removing at least a portion of the water from the aqueous composition such that, at the end of the polymerization and/or condensation reaction, the water content of the aqueous composition is as described above. In some embodiments, the method includes removing at least a portion of the water from the aqueous composition such that, at the start of the polymerization and/or condensation reaction, the average water content of the aqueous composition is as described above. In some embodiments, the method includes removing at least a portion of the water from the aqueous composition such that the average water content of the aqueous composition at the beginning and end of the polymerization and/or condensation reaction is within the range described above. In some embodiments, the method includes removing at least a portion of the water from the aqueous composition such that, throughout the polymerization and/or condensation reaction, the water content of the aqueous composition is within the range described above.

In some embodiments, the method described herein includes adding at least a portion of water to the aqueous composition such that the water content of the aqueous composition is from about 1 to 20%, about 1 to 18%, about 1 to 16%, about 1 to 14%, about 1 to 12%, about 1 to 10%, about 1 to 8%, about 2 to 16%, about 2 to 14%, about 2 to 12%, about 2 to 10%, about 2 to 8%, about 2 to 6%, about 4 to 16%, about 4 to 14%, about 4 to 12%, about 4 to 10%, about 4 to 8%, about 6 to 16%, about 6 to 12%, about 6 to 10%, or about 6 to 8% by weight. In some embodiments, the method includes adding at least a portion of water to the aqueous composition such that the water content of the aqueous composition is from about 2 to 10%, about 2 to 8%, or about 4 to 8% by weight. In some embodiments, the method includes adding at least a portion of water to the aqueous composition such that the water content of the aqueous composition is about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9% or about 10% of the total weight. In some embodiments, the method includes adding at least a portion of water to the aqueous composition such that the water content of the aqueous composition is from about 4 to 8% by weight. In some embodiments, the method includes adding at least a portion of water to the aqueous composition such that, at the end of the polymerization and/or condensation reaction, the water content of the aqueous composition is as described above. In some embodiments, the method includes adding at least a portion of water to the aqueous composition such that, at the start of the polymerization and/or condensation reaction, the water content of the aqueous composition is as described above. In some embodiments, the method includes adding at least a portion of water to the aqueous composition such that the average water content of the aqueous composition at the beginning and end of the polymerization and/or condensation reaction is within the range described above. In some embodiments, the method includes adding at least a portion of water to the aqueous composition such that, throughout the polymerization and/or condensation reaction, the water content of the aqueous composition remains within the range described above.

In some embodiments, the degree of polymerization of the oligosaccharides and/or the amount and type of anhydrosubunits in the oligosaccharide preparation can be controlled by adjusting or controlling the water content present in the aqueous composition throughout the manufacturing process. For example, in some embodiments, the degree of oligosaccharide polymerization and the number of anhydrosubunits are increased by decreasing the water content.

Accordingly, in some embodiments, the method described herein includes in-process control (IPC) of water content, which may include monitoring water content, maintaining water content, increasing water content, decreasing water content, or any combination thereof. In some embodiments, the IPC process includes maintaining the water content while the aqueous composition is heated to the temperature described herein. In some embodiments, the method includes maintaining a water content for a time sufficient to induce polymerization. In some embodiments, the method includes maintaining the water content within a disclosed range by either adding water or removing water from the aqueous composition, or both. In some embodiments, the method includes maintaining the water content within a disclosed range by distillation. In some embodiments, the method includes maintaining the water content within a disclosed range using vacuum distillation. In some embodiments, the method includes maintaining the water content within the disclosed range by distillation at atmospheric pressure.

In some embodiments, the water content of the aqueous composition is maintained at

- 32 044359 range from about 1 to 20%, from about 1 to 18%, from about 1 to 16%, from about 1 to 14%, from about 1 to 12%, from about 1 to 10%, from about 1 to 8%, about 2 to 16%, about 2 to 14%, about 2 to 12%, about 2 to 10%, about 2 to 8%, about 2 to 6%, about 4 to 16% , about 4 to 14%, about 4 to 12%, about 4 to 10%, about 4 to 8%, about 6 to 16%, about 6 to 12%, about 6 to 10% or approximately 6 to 8% of the total mass. In some embodiments, the water content of the aqueous composition is maintained in the range of about 2 to 10%, about 2 to 8%, or about 4 to 8% by weight. In some embodiments, the water content of the aqueous composition is maintained in the range of from about 2 to 8% by weight.

The water content of an aqueous composition can be determined by a variety of analytical methods and instruments. In some embodiments, water content is determined by evaporation method (eg, loss on drying method), distillation method, or chemical reaction method (eg, Karl Fischer titration). In some embodiments, the water content is determined by an analytical instrument, such as a moisture analyzer. In some embodiments, water content is determined by Karl Fischer titration.

In some embodiments, the water content of the aqueous composition is measured during the reaction and used to implement in-process water content control (IPC). In some embodiments, the water content of the reaction is measured by Karl Fischer titration, IR spectroscopy, NIR spectroscopy, conductivity, viscosity, density, stirring torque, or stirring energy. In some embodiments, measurement of the water content of the reaction is used to control a device that actively controls the water content of the reaction, such as a water addition pump or flow valve.

Without wishing to be bound by theory, it is believed that the water content during the sugar polymerization and/or condensation reaction may influence the level of anhydrous subunits in the oligosaccharide preparation described herein. For example, as shown in FIG. 34, in some embodiments, higher water content correlates with lower levels of anhydrous subunits. In some embodiments, a lower reaction temperature may correlate with a lower level of anhydrous subunits.

E. Temperature

In some embodiments, the degree of polymerization of the oligosaccharides and/or the number and type of anhydrosubunits in the oligosaccharide preparation can be controlled by adjusting the temperature to which the aqueous composition is heated. In some embodiments, a method for producing an oligosaccharide preparation described herein comprises heating the aqueous composition to a temperature of about 80 to 250°C, about 90 to 200°C, about 100 to 200°C, about 100 to 180°C, about 110 to 170°C, about 120 to 160°C, about 130 to 150°C, or about 135 to 145°C. In some embodiments, a method for producing an oligosaccharide preparation includes heating the aqueous composition to a temperature of about 100 to 200°C, about 100 to 180°C, about 110 to 170°C, about 120 to 160°C, about 130 to 150°C or approximately 135 to 145°C. In some embodiments, a method for producing an oligosaccharide preparation includes heating the aqueous composition to a temperature of about 135 to 145°C. In other embodiments, a method for producing an oligosaccharide preparation includes heating the aqueous composition to a temperature of about 125 to 135°C.

F. Reaction time

In some embodiments, a method for producing an oligosaccharide preparation described herein comprises heating the aqueous composition for a sufficient time. In some embodiments, the degree of polymerization of oligosaccharides prepared in accordance with the methods described herein can be adjusted by reaction time.

In some embodiments, sufficient time is described in terms of hours. For example, in some embodiments, a sufficient time is at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours , at least 7 hours, at least 8 hours, at least 9 hours, or at least 10 hours. In some embodiments, a sufficient time is about 1 to 24 hours, about 1 to 16 hours, about 1 to 8 hours, about 1 to 4 hours, about 1 to 3 hours, about 1 to 2 hours, about 2 to 12 hours, about 2 to 10 hours, about 2 to 8 hours, about 2 to 6 hours, about 2 to 4 hours, about 3 to 8 hours, about 3 to 6 hours, about 3 to 5 hours, or about 3 to 4 hours.

In other embodiments, sufficient time is determined by measuring one or more chemical or physical properties of the oligosaccharide preparation, such as water content, viscosity, molecular weight, anhydrous subunit content, and/or degree of polymerization distribution.

In some embodiments, the molecular weight of the oligosaccharide drug is controlled during polymerization. In some embodiments, the method includes heating the aqueous composition for a time sufficient for the aqueous composition to reach the media.

- 33 044359 non-numerical molecular weight or weight average molecular weight, as described in this application. In some embodiments, the method includes heating the aqueous composition for a time sufficient to achieve a number average molecular weight of the aqueous composition in the range of about 300 to 5000 g/mol, about 500 to 5000 g/mol, about 700 to 5000 g/mol, about 500 to 2000 g/mol, about 700 to 2000 g/mol, about 700 to 1500 g/mol, about 300 to 1500 g/mol, about 300 to 2000 g/mol, about 400 to 1000 g/mol, about 400 to 900 g/mol, about 400 to 800 g/mol, about 500 to 900 g/mol, or about 500 to 800 g/mol. In some embodiments, the method includes heating the aqueous composition for a time sufficient to achieve the aqueous composition having a number average molecular weight of about 500 to 2000 g/mol. In some embodiments, the method includes heating the aqueous composition for a time sufficient to achieve a weight average molecular weight of the aqueous composition in the range of about 300 to 5000 g/mol, about 500 to 5000 g/mol, about 700 to 5000 g/mol, about 500 to 2000 g/mol, about 700 to 2000 g/mol, about 700 to 1500 g/mol, about 300 to 1500 g/mol, about 300 to 2000 g/mol, about 400 to 1300 g/mol, about 400 to 1200 g/mol, about 400 to 1100 g/mol, about 500 to 1300 g/mol, about 500 to 1200 g/mol, about 500 to 1100 g/mol, about 600 to 1300 g/mol, about 600 to 1200 g/mol, or about 600 to 1100 g/mol. In some embodiments, the method includes heating the aqueous composition for a time sufficient to achieve a weight average molecular weight of the aqueous composition from about 700 to 3000 g/mol.

In some embodiments, sufficient time is the time required for the aqueous composition to reach reaction equilibrium at the appropriate reaction temperature. Accordingly, in some embodiments, the method includes heating the aqueous composition for a time sufficient for the aqueous composition to reach equilibrium. For example, in some embodiments, equilibrium is determined by measuring the molecular weight, viscosity, or DP distribution of the aqueous composition.

In some embodiments, equilibrium is determined by measuring the number average or weight average molecular weight of the aqueous composition. In some embodiments, the equilibrium is determined by the number-average or weight-average molecular weight of the aqueous composition, which remains substantially unchanged over time. In some embodiments, equilibrium is determined by a change in the number-average or weight-average molecular weight of the aqueous composition that is less than a certain percentage over a period of time. In some embodiments, the molecular weight of the aqueous composition is measured using HPLC or SEC.

In some embodiments, equilibrium is determined by a change in the number average or weight average molecular weight of the aqueous composition of less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% over a period of time. In some embodiments, equilibrium is determined by changing the number average or weight average molecular weight of the aqueous composition over 3, 2, 1 hour, 30, 20, or 10 minutes. In some embodiments, equilibrium is determined by a change in the weight average molecular weight of the aqueous composition by less than 15% over a period of 1 hour.

In some embodiments, equilibrium is determined by measuring the viscosity of the aqueous composition. In some embodiments, equilibrium is determined by the viscosity of the aqueous composition, which remains substantially unchanged over time. In some embodiments, equilibrium is determined by a change in viscosity of the aqueous composition that is less than a certain percentage over a period of time. In some embodiments, the viscosity of the aqueous composition is measured with a viscometer or rheometer.

In some embodiments, equilibrium is determined by a change in viscosity of the aqueous composition of less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% over a period of time. In some embodiments, equilibrium is determined by the change in viscosity of the aqueous composition over 3, 2, 1 hour, 30, 20, or 10 minutes. In some embodiments, equilibrium is determined by a change in the viscosity of the aqueous composition of less than 15% over a period of 1 hour.

In some embodiments, equilibrium is determined by measuring the DP distribution of the aqueous composition. In some embodiments, equilibrium is determined by the DP distribution of the aqueous composition, which remains substantially unchanged over time. In some embodiments, the change in DP distribution of an aqueous composition is determined by calculating _{Kt =} [or _t ][n ₂ o] series Km, where [ο₽>η-ι][ο₽ΐ]' where [H2O] is the molar concentration of water (mol/L), and [DP1], [DPm-1] and [DPm] are the molar concentrations of oligosaccharides (mol/L) in DP1, DPm-1 and DPm fractions, respectively. For example, K2 is equal to [DP2][H2O]/[DP1][DP1] according to the above formula. In some embodiments, m is an integer greater than 1 and less than n. In other embodiments, m is equal to n. In some embodiments, m is an integer greater than 1 and equal to or less than n. In some embodiments,

- 34 044359 m is equal to 2, 3, 4, 5, 6, 7, 8, 9 or 10.

In some embodiments, the concentration of oligosaccharides in the DP1, DPm-1, and DPm fractions is determined using SEC, HPLC, PFP, A4F, mass spectrometry, or any other suitable method. In some embodiments, the concentration of oligosaccharides in the DP1, DPm-1, and DPm fractions is determined using SEC, such as GPC. In some embodiments, the concentration of oligosaccharides in the DP1, DPm-1, and DPm fractions is determined by mass spectrometry, such as GC-MS, LC-MS/MS, and MALDI-MS. In some embodiments, the concentration of oligosaccharides in the DP1, DPm-1, and DPm fractions is determined by HPLC. In some embodiments, the water concentration is determined by evaporation method (eg, loss on drying method), distillation method, or chemical reaction method (eg, Karl Fischer titration). In some embodiments, the water concentration is determined by any suitable analytical instrument, such as a moisture analyzer.

In some embodiments, the method includes calculating a series of at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 30, at least 40 or at least 50 Km values. In some embodiments, the method includes calculating a series of at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or at least 15 Km values. In some embodiments, the method includes calculating about 3, 4, 5, 6, 7, 8, 9, 10, or 15 Km values. In some embodiments, the method includes calculating K2 to K4, K2 to K5, K2 to K6, K2 to K7, K2 to K8, K2 to K9, K2 to K10, K2 to K11, K2 to K12, K2 to K13, K2 to K14, K2 to K15, K3 to K5, K3 to K6, K3 to K7, K3 to K8, K3 to K9, K3 to K10, K3 to K11, K3 to K12, K3 to K13, K3 to K14, or K3 to K15. In some embodiments, the method includes calculating K2 to K4 or K3 to K5.

In some embodiments, the Km value depends on temperature, water concentration, and/or the amount and type of feed sugars. In some embodiments, Km is from about 0.1 to 100, about 0.1 to 90, about 0.1 to 80, about 0.1 to 70, about 0.1 to 60, about 0. 1 to 50, about 0.1 to 40, about 0.1 to 30, about 0.1 to 25, about 0.1 to 20, or about 0.1 to 15. In some embodiments, Km is about 1 to 100, about 1 to 90, about 1 to 80, about 1 to 70, about 1 to 60, about 1 to 50, about 1 to 40, about 1 to 30, about 1 to 25, about 1 to 20, about 1 to 15, about 1 to 10, about 5 to 50, about 5 to 40, about 5 to 30, about 5 to 20, about 5 to 15, or about 5 to 10. In some embodiments, Km is from about 1 to 15, or about 5 to 15.

In some embodiments, the mean, standard deviation, and/or relative standard deviation are determined for a series of calculated Km. In this context, relative standard deviation is expressed as a percentage and is obtained by multiplying the standard deviation by 100 and dividing this product by the mean.

In some embodiments, equilibrium is defined by a relative standard deviation of the series Km less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5% , less than 4%, less than 3%, less than 2% or less than 1%. In some embodiments, equilibrium is defined by a relative standard deviation of the Km series of less than 15%, less than 10%, or less than 5%.

G. Post-reaction steps

In some embodiments, the method of producing oligosaccharide preparations described herein further includes one or more additional processing steps after heating the aqueous composition at a temperature and for a sufficient time. In some embodiments, additional processing steps include, for example, separation (such as chromatographic separation), dilution, concentration, drying, filtration, demineralization, extraction, decolorization, or any combination thereof. For example, in some embodiments, the method includes a dilution step and a decolorization step. In some embodiments, the method includes a filtration step and a drying step.

In some embodiments, the method includes a dilution step in which water is added to the oligosaccharide preparation to produce a syrup of the oligosaccharide preparation. In some embodiments, the concentration of oligosaccharide drug in the syrup is about 5 to 80%, about 10 to 70%, about 10 to 60%, about 10 to 50%, about 10 to 40%, about 10 to 30 % or about 15 to 25%. In other embodiments, the method does not include a dilution step, but rather the oligosaccharide preparation is allowed to solidify. In some embodiments, the method includes a filtration step. In some embodiments, the method includes recycling the catalyst by filtration.

In some embodiments, the described method includes a decolorization step. In some embodiments, the oligosaccharide preparation may be subjected to a decolorization step using any method known in the art, including, for example, treatment with an absorbent, activated carbon, chromatography (eg, using an ion exchange resin), hydrogenation, and/or filtration (eg microfiltration).

In some embodiments, the oligosaccharide preparation is contacted with a material to remove salts, minerals, and/or other ionic species. In some embodiments, the oligosaccharide drug is passed through a pair of anion/cation exchange columns. In one embodiment, the anion exchange column contains a weak base exchange resin in the hydroxide form, and the cation exchange column contains a strong acid exchange resin in the protonated form.

In some embodiments, the method includes a concentration step. In some embodiments, the concentration step produces an increased concentration of the oligosaccharide drug. For example, in some embodiments, the concentration step includes evaporation (eg, vacuum evaporation), drying (eg, lyophilization and spray drying), or any combination thereof.

In some embodiments, the method includes a separation step in which at least a portion of the oligosaccharide drug is separated. In some embodiments, the isolation step includes crystallization, precipitation, filtration (eg, vacuum filtration), and centrifugation, or any combination thereof.

In some embodiments, the method includes a separation step. In some embodiments, the separation step includes separating at least a portion of the oligosaccharide preparation from at least a portion of the catalyst, at least a portion of the unreacted feed sugars, or both. In some embodiments, the separation step includes filtration, chromatography, differential solubility, precipitation, extraction, or centrifugation.

H. Reactors

The methods described herein may include the use of one or more reactors suitable for condensing the sugar, taking into account reaction temperature, pH, pressure and other factors. In some embodiments, one or more suitable reactors include a fed-batch reactor, a stirred batch reactor, a continuous stirred flow reactor, a continuous plug flow reactor, an attrition reactor, or an electromagnetic field induced stirred reactor. In some embodiments, the one or more suitable reactors include the reactor described in Ryu, S. K., and Lee, J. M., Bioconversion of waste cellulose by using an attrition bioreactor, Biotechnol. Bioeng. 25: 53-65(1983); Gusakov, A. V., and Sinitsyn, A. P., Kinetics of the enzymatic hydrolysis of cellulose: 1. A mathematical model for a batch reactor process, Enz. Microb. TechnoL, 7: 346-352 (1985); Gusakov, A. V., Sinitsyn, A. P., Davydkin, I. Y., Davydkin, V. Y., Protas, O. V., Enhancement of enzymatic cellulose hydrolysis using a novel type of bioreactor with intensive stirring induced by electromagnetic field, Appl. Biochem. Biotechnol., 56: 141-153(1996); or Fernanda de Castilhos Corazza, Flavio Faria de Moraes, Gisella Maria Zanin and Ivo Neitzel, Optimal control in fed-batch reactor for the cellobiose hydrolysis, Acta Scientiarum. Technology, 25: 33-38 (2003).

In some embodiments, one or more suitable reactors include fluidized bed, upflow blanket, immobilization, or extruder type reactors for hydrolysis and/or fermentation. In some embodiments, the one or more suitable reactors include an open reactor, a closed reactor, or both. In some embodiments, when the method involves a continuous process, one or more suitable reactors may include a continuous mixer, such as a screw mixer.

Process

In some embodiments, a method for producing oligosaccharide preparations described herein comprises a batch process, a continuous process, or both. In some embodiments, a method for producing an oligosaccharide drug includes a batch process. For example, in some embodiments of a batch process, production of subsequent batches of the oligosaccharide drug is not started until the current batch is completed. In some embodiments, during a batch process, all or a substantial amount of the oligosaccharide drug is removed from the reactor. In some embodiments, during a batch process, all feed sugars and catalyst are combined in a reactor before the aqueous composition is heated to the described temperature or before polymerization is initiated. In some embodiments, during a batch process, feed sugars are added before, after, or simultaneously with the addition of the catalyst.

In some embodiments, the batch process is a fed-batch process where all feed sugars are not added to the reactor at the same time. In some fed-batch embodiments, at least a portion of the feed sugars are added to the reactor during polymerization or after the aqueous composition has been heated to the described temperature. In some fed-batch embodiments, at least 10%, 20%, 30%, 40%, 50%, or 60% by weight of the feed sugars are added to the reactor during polymerization or after the aqueous composition is heated to the described temperature.

In some embodiments, the method of producing an oligosaccharide drug is a continuous process. For example, in some embodiments of a continuous process, the reactor contents flow continuously through the reactor. In some embodiments, the combination of feed sugars with a catalyst and removal of at least a portion of the oligosaccharide drug are performed simultaneously.

In some embodiments, a method for producing an oligosaccharide drug includes a one-pot and a multi-pot process. For example, in some embodiments of a one-pot process, the polymerization is carried out in a single reactor. As another example, in some embodiments of a multi-reactor process, polymerization is carried out in more than one reactor. In some embodiments of a multi-reactor process, the method includes 2, 3, or more reactors. In some embodiments of a multi-reactor process, the method includes a combining step in which polymerization products from two or more reactors are combined.

I. Process

In some embodiments, a method for producing oligosaccharide preparations comprises a batch process, a continuous process, or both. In some embodiments, a method for producing an oligosaccharide drug includes a batch process. For example, in some embodiments of a batch process, production of subsequent batches of the oligosaccharide drug is not started until the current batch is completed. In some embodiments, during a batch process, all or a substantial amount of the oligosaccharide drug is removed from the reactor. In some embodiments, during a batch process, all feed sugars and catalyst are combined in a reactor before the aqueous composition is heated to the described temperature or before polymerization is initiated. In some embodiments, during a batch process, feed sugars are added before, after, or simultaneously with the addition of the catalyst.

In some embodiments, the batch process is a fed-batch process where all feed sugars are not added to the reactor at the same time. In some fed-batch embodiments, at least a portion of the feed sugars are added to the reactor during polymerization or after the aqueous composition has been heated to the described temperature. In some fed-batch embodiments, at least 10%, 20%, 30%, 40%, 50%, or 60% by weight of the feed sugars are added to the reactor during polymerization or after the aqueous composition is heated to the described temperature.

In some embodiments, the method of producing an oligosaccharide drug is a continuous process. For example, in some embodiments of a continuous process, the contents of the reactor flow continuously through the reactor. In some embodiments, the combination of feed sugars with a catalyst and removal of at least a portion of the oligosaccharide drug are performed simultaneously.

In some embodiments, a method for producing an oligosaccharide drug includes a one-pot and a multi-pot process. For example, in some embodiments of a one-pot process, the polymerization is carried out in a single reactor. As another example, in some embodiments of a multi-reactor process, polymerization is carried out in more than one reactor. In some embodiments of a multi-reactor process, the method includes 2, 3, or more reactors. In some embodiments of a multi-reactor process, the method includes a combining step in which polymerization products from two or more reactors are combined.

IV. Nutritional compositions including oligosaccharide preparations

This application provides nutritional compositions containing an oligosaccharide preparation. In some embodiments, this application provides nutritional compositions containing a disclosed oligosaccharide drug, wherein the presence and/or concentration of the oligosaccharide drug in the nutritional compositions can be selectively determined and/or detected. Oligosaccharide drugs that exhibit complex functional modulation of the microbial community can be important components of nutritional formulations. Thus, the presence and/or concentration of the oligosaccharide drug in nutritional compositions may be one of the factors that needs to be measured during quality control and production of nutritional compositions. Accordingly, the nutritional compositions of the invention are advantageous for quality control and manufacturing purposes because the presence and/or concentration of the oligosaccharide drug can be selectively determined and/or detected. For example, in some embodiments, the presence and concentration of an oligosaccharide drug can be determined and/or detected by measuring the signal associated with oligosaccharides containing anhydrosaccharide units.

In some embodiments, the nutritional composition is an animal feed composition. In some embodiments, the nutritional composition includes a base nutritional composition.

A. Basic Nutritional Compositions

In some embodiments, the primary nutritional composition contains a carbohydrate source other than the oligosaccharide drug. For example, in some embodiments, the base nutritional composition includes a natural carbohydrate source such as starch and plant fiber. In some embodiments, the base nutritional composition comprises starch. In some embodiments, the base nutritional composition comprises plant fibers.

In some embodiments, the base nutritional composition contains one or more carbohydrate sources that are derived from seeds, roots, tubers, corn, tapioca, arrowroot, wheat, rice, potatoes, sweet potatoes, sago, beans (e.g., broad beans, lentils, beans mung beans, peas and chickpeas), corn, cassava or other starchy foods (e.g. acorns, arrowroot, arracachis, bananas, barley, breadfruit, buckwheat, canna, taro, japonica, pueraria, xanthosoma, millet, oats, oxalis) , Polynesian arrowroot, sorghum, rye, taro, chestnut, water chestnut and yam).

In some embodiments, the base nutritional composition contains one or more carbohydrate sources that are derived from legumes (e.g., peas, soybeans, lupine, green beans, and other beans), oats, rye, chia, barley, fruits (e.g., figs, avocado , plums, prunes, berries, bananas, apple, quince and pear peels), vegetables (such as broccoli, carrots, cauliflower, zucchini, celery, nopal and Jerusalem artichoke), root tubers, root vegetables (such as sweet potatoes and onions), psyllium husks, seeds (eg flax seeds), nuts (eg almonds), whole grains, wheat, corn bran, lignans, or any combination thereof. In some embodiments, the composition includes one or more plant fibers derived from wheat bran, sugar beet pulp, cottonseed, soybean husk, or any combination thereof.

In some embodiments, the base nutritional composition contains less than 500 ppm, less than 400 ppm, less than 300 ppm, less than 200 ppm, less than 100 ppm, less than 50 ppm, less than 10 ppm, less than 5 ppm or less than 1 ppm anhydrosubunits or oligosaccharides containing anhydrosubunits. In some embodiments, the basic nutritional composition contains less than 50 ppm, less than 10 ppm, less than 5 ppm, or less than 1 ppm anhydrosubunits or oligosaccharides containing anhydrosubunits. In some embodiments, the basic nutritional composition is substantially free of anhydrous subunits.

In some embodiments, the base nutritional composition lacks detectable levels of anhydrous subunits. Depending on the detection or determination methods, levels of anhydrosubunits below a certain threshold may not be detectable. For example, in some embodiments, the detectable level of anhydrosubunits may be at least 1000 ppm, at least 500 ppm, at least 400 ppm, at least 300 ppm, at least at least 200 ppm, at least 100 ppm, at least 50 ppm, at least 10 ppm, at least 5 ppm or at least 1 ppm million anhydrosubunits or oligosaccharides containing anhydrosubunits in the basic nutritional composition.

In some embodiments, the base nutritional composition contains a plurality of oligosaccharides. In some embodiments, the base nutritional composition includes a glycosidic linkage type distribution that is different from the oligosaccharide preparation. For example, in some embodiments, the base nutritional composition contains a higher percentage of α-(1,4) glycosidic linkages than the oligosaccharide preparation. In some embodiments, glycosidic linkages, such as α-(1,4) glycosidic linkages in basic nutritional compositions, are cleaved by one or more enzymes. In some embodiments, the glycosidic bonds in the base nutritional composition are more easily cleaved and/or hydrolyzed than the glycosidic bonds in the oligosaccharide preparation.

In some embodiments, the level of α-(1,2) glycosidic bond, α-(1,3) glycosidic bond, α-(1,6) glycosidic bond, β-(1,2) glycosidic bond, β-(1, 3) glycosidic bond, β-(1,4) glycosidic bond or β-(1,6) glycosidic bond in the main nutritional composition by at least 2%, at least 3%, at least 4%, according to at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least at least 12%, at least 13%, at least 14%, or at least 15% below the level of the corresponding glycosidic bond in the oligosaccharide preparation. In some embodiments, the level of α-(1,2) glycosidic bond, α-(1,3) glycosidic bond, α-(1,6) glycosidic bond, β-(1,2) glycosidic bond, β-(1, 3) a glycosidic bond, a β-(1,4) glycosidic bond, or a β-(1,6) glycosidic bond in the basic nutritional composition that is at least 10% lower than the level of the corresponding glycosidic bond in the oligosaccharide preparation.

In some embodiments, the level of α-(1,4) glycosidic linkage in the base nutritional composition is at least 50%, at least 40%, at least 35%, at least 30%, at least 25%, at least 20%, at least 15%, at least 10%, at least 5% or at least 2% higher than the level of the α-(1,4) glycosidic linkage in the oligosaccharide drug. In some embodiments, the level of α-(1,4) glycosidic linkage in the base nutritional composition is at least 10% higher than the level of α-(1,4) glycosidic linkage in the oligosaccharide preparation.

B. Animal feed composition

Depending on the type and age of the animal, the nutritional composition may contain the oligosaccharide preparation and the main nutritional composition in different ratios. For example, the oligosaccharide preparation can be combined with the basic nutritional composition in various ratios suitable for the type and age of the animal. In some embodiments, the oligosaccharide drug is present in the nutritional composition at a concentration of about 1 to 10,000 ppm, about 1 to 5,000 ppm, about 1 to 3,000 ppm, about 1 to 2,000 ppm ppm, about 1 to 1500 ppm, about 1 to 1000 ppm, about 1 to 500 ppm, about 1 to 250 ppm, about 1 to 100 ppm ppm, about 10 to 5000 ppm, about 10 to 3000 ppm, about 10 to 2000 ppm, about 10 to 1500 ppm, about 10 to 1000 ppm ppm, about 10 to 500 ppm, about 10 to 250 ppm, about 10 to 100 ppm, about 50 to 5000 ppm, about 50 to 3000 ppm ppm, about 50 to 2000 ppm, about 50 to 1500 ppm, about 50 to 1000 ppm, about 50 to 500 ppm, about 50 to 250 ppm ppm, about 50 to 100 ppm, about 100 to 5000 ppm, about 100 to 3000 ppm, about 100 to 2000 ppm, about 100 to 1500 ppm ppm, about 100 to 1000 ppm, about 100 to 500 ppm, about 100 to 400 ppm, about 100 to 300 ppm, about 100 to 200 ppm ppm, about 200 to 5000 ppm, about 200 to 3000 ppm, about 200 to 2500 ppm, about 200 to 2000 ppm, about 200 to 1500 ppm ppm, about 200 to 1000 ppm, about 200 to 500 ppm, about 500 to 5000 ppm, about 500 to 3000 ppm, about 500 to 2500 ppm ppm, about 500 to 2000 ppm, about 500 to 1500 ppm, or about 500 to 1000 ppm. In some embodiments, the oligosaccharide drug is present in the nutritional composition at a concentration of about 1 to 5000 ppm, about 1 to 1000 ppm, about 1 to 500 ppm, about 10 to 5000 ppm ppm, about 10 to 2000 ppm, about 10 to 1000 ppm, about 10 to 500 ppm, about 10 to 250 ppm, about 10 to 100 ppm ./million, from about 50 to 5000 ppm, from about 50 to 2000 ppm, from about 50 to 1000 ppm, from about 50 to 500 ppm, from about 50 to 250 ppm or about 50 to 100 ppm. In some embodiments, the oligosaccharide drug is present in the nutritional composition at a concentration of about 1 to 5000 ppm, about 10 to 1000 ppm, about 10 to 500 ppm, or about 50 to 500 ppm million

In some embodiments, the oligosaccharide drug is present in the nutritional composition at a concentration of greater than 10 ppm, greater than 50 ppm, greater than 100 ppm, greater than 200 ppm, greater than 300 ppm, greater than 400 ppm ./million, more than 500 ppm, more than 600 ppm, more than 1000 ppm or more than 2000 ppm. In some embodiments, the oligosaccharide drug is present in the nutritional composition at a concentration of greater than 10 ppm, greater than 50 ppm, greater than 100 ppm, greater than 200 ppm, or greater than 500 ppm.

In some embodiments, depending on the type and age of the animal, the nutritional composition may further include proteins, minerals (such as copper, calcium and zinc), salts, essential amino acids, vitamins and/or antibiotics.

The present application also provides a method of administering to an animal a nutritional composition containing a basic nutritional composition and the described oligosaccharide preparation. In some embodiments, the animal is selected from cattle (eg, beef and dairy cattle), swine, aquatic animals, poultry, and humans. In some embodiments, the animal is a pig, such as sows, piglets, and hogs. In other embodiments, the animal is a poultry such as chicken, duck, turkey, goose, quail and chicken. In some embodiments, the poultry is a broiler, breeder, or layer. In some embodiments, the animal is an aquatic animal such as salmon, catfish, grouper, eel, tilapia, flounder, shrimp, and crab. In some embodiments, the nutritional composition is provided to the animal in dry form, liquid form, paste form, or a combination thereof. In some embodiments, the form of application, feeding rate, and feeding regimen may vary depending on the type and age of the animal.

- 39 044359

C. Methods for producing nutritional compositions

Provided herein are methods for producing a nutritional composition comprising combining an oligosaccharide preparation with a base nutritional composition. In some embodiments, the oligosaccharide preparation includes oligosaccharides containing anhydrosubunits. In some embodiments, the oligosaccharide preparation includes a glycosidic linkage type distribution that is different from that of the base nutritional composition.

In some embodiments, the oligosaccharide drug is a synthetic oligosaccharide drug. In some embodiments, the synthetic oligosaccharide preparation contains at least n oligosaccharide fractions, each having a different degree of polymerization selected from 1 to n (fractions DP1 to DPn). In some embodiments, n is an integer equal to 2 or more. In some embodiments, n is an integer greater than 2. In some embodiments, n is an integer equal to 3 or greater. In some embodiments, n is an integer ranging from 1 to 100, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or 50. In some embodiments, each of the DP1DPn fractions comprises from 0.1 to 90% anhydrosubunit-containing oligosaccharides, by relative abundance , measured using mass spectrometry. In some embodiments, each of the oligosaccharide preparation fractions DP1 and DP2 independently comprises from about 0.1 to 15% or about 0.5 to 10% anhydrosubunit-containing oligosaccharides, as measured by relative abundance by mass spectrometry. In some embodiments, each of the oligosaccharide preparation fractions DP1 and DP2 independently comprises oligosaccharides containing anhydrosubunits ranging from about 0.1%, 0.5%, 0.6%, 0.7%, 0.8%, 0. 9%, 1%, 1.1%, 1.2%, 1.3%, 1.4% or 1.5% to 8%, 9%, 10%, 11%, 12%, 15% or 20 % by relative abundance measured by mass spectrometry. In some embodiments, the relative abundance of oligosaccharides in each of the n fractions decreases monotonically with the degree of polymerization. In some embodiments, the relative abundance of oligosaccharides in at least 5, 10, 20, or 30 DP fractions decreases monotonically with degree of polymerization.

In some embodiments, a method for producing a nutritional composition includes mixing an oligosaccharide preparation with a base nutritional composition. For example, in some embodiments, mixing may be accomplished using a commercial mixer and/or a mixer such as a drum mixer, a double cone mixer, a ribbon mixer, a V mixer, a shear mixer, and a paddle mixer.

In some embodiments, the method of producing a nutritional composition further includes a quality control step described herein. In some embodiments, the quality control step described herein includes determining a signal level in a sample of the nutritional composition and calculating the concentration of an oligosaccharide drug in the nutritional composition based on the signal level. In some embodiments, the quality control step described herein includes detecting a signal in a sample of the nutritional composition using analytical instruments, and accepting or rejecting a batch of the nutritional composition based on the presence or absence of the signal. In some embodiments, the quality control step described herein includes detecting by analytical instruments the presence or absence of a first signal in a first sample of the nutritional composition and a second signal in a second sample of the nutritional composition, and comparing the first signal and the second signal. In some embodiments, the signal, the first signal and/or the second signal (i) indicates oligosaccharides containing one or more anhydrosubunits; (ii) is associated with the distribution of oligosaccharides according to the degree of polymerization (DP); or (iii) associated with α-(1,2) glycosidic bonds, α-(1,3) glycosidic bonds, α-(1,6) glycosidic bonds, β-(1,2) glycosidic bonds, β-(1 ,3) glycosidic bonds, β-(1,4) glycosidic bonds or β-(1,6) glycosidic bonds of oligosaccharides.

Additionally, in some embodiments, a method for producing a nutritional composition includes, after performing a quality control step, further mixing the oligosaccharide drug with the base nutritional composition, adjusting the level of the oligosaccharide drug, or a combination thereof. In some embodiments, adjusting the level of the oligosaccharide drug includes adding an additional oligosaccharide drug to the nutritional composition or removing a portion of the oligosaccharide drug from the nutritional composition. In some embodiments, adjusting the level of the oligosaccharide drug includes adding an additional primary nutritional composition to the nutritional composition or removing a portion of the primary nutritional composition from the nutritional composition. In some embodiments, adjusting the level of the oligosaccharide drug includes adding an additional oligosaccharide drug to the nutritional composition.

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D. Animal feed premix

In some embodiments, the nutritional composition includes an animal feed premix containing a disclosed oligosaccharide preparation.

In some embodiments, the animal food premix comprises a carrier material that can be combined with an oligosaccharide drug to form the animal food premix. In some embodiments, the carrier material can be any material in dry or liquid form that is suitable for combination with an oligosaccharide drug in a nutritional composition. In some embodiments, the carrier material includes grain fermentation solids, clay, vermiculite, diatomaceous earth, hulls such as milled rice hulls and milled oat hulls, silica such as feed grade silica gel and feed grade colloidal silica, corn, such as corn gluten, corn gluten meal and ground corn, or any combination thereof. In some embodiments, the carrier material is ground corn. In other embodiments, the carrier material is milled rice hulls or milled oat hulls.

In some embodiments, the animal feed premix is prepared by combining a carrier material with an oligosaccharide drug, both in dry form. In some embodiments, the animal feed premix is prepared by combining a carrier material with an oligosaccharide preparation; one of which is in dry form. In some embodiments, the animal feed premix is prepared by combining a carrier material with an oligosaccharide drug, both of which are in liquid form. For example, in some embodiments, an oligosaccharide preparation in liquid form refers to an oligosaccharide in solution, such as an aqueous solution of oligosaccharides, such as a syrup.

In some embodiments, the animal feed premix is prepared by combining a carrier material with a syrup containing an oligosaccharide drug. In some embodiments, the concentration of oligosaccharide drug in the syrup is at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70 %, at least 75% or at least 80% by weight. In some embodiments, the concentration of oligosaccharide drug in the syrup is from about 40 to 80%, from 50 to 75%, or from 60 to 70% by weight.

In some embodiments, the pet food premix is in the form of a powder (eg, a pourable powder), slurry, slurry, granules, or liquid. In some embodiments, the animal food premix has a moisture content of less than 40%, 30%, 20%, 15%, 10%, or 5% by weight. In some embodiments, the pet food premix has a moisture content of less than 10% or 5% by weight. In some embodiments, the animal food premix has a moisture content of greater than 5%, 10%, 15%, 20%, 25%, or 30% by weight. In additional embodiments, the moisture content of the animal feed premix is adjusted to any of the described ranges. For example, in some embodiments, the pet food premix is dried to increase the moisture content within the described range.

In some embodiments, depending on the specific application, the animal feed premix contains varying levels of the oligosaccharide drug. In some embodiments, the animal food premix contains at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% , 95% or 99% oligosaccharide preparation by dry matter weight. In some embodiments, the animal food premix contains at most 50%, 60%, 70%, 80%, 90%, 95%, or 99% oligosaccharide drug by dry matter weight.

In some embodiments, the animal food premix or carrier material further comprises other animal food such as minerals, fats and proteins. In some embodiments, the animal feed carrier material or premix contains copper, zinc, or both. In some embodiments, the carrier material or animal feed premix contains an ionophore or other coccidiostat. In some embodiments, the animal feed carrier material or premix contains an antibiotic. In some embodiments, the carrier material includes a source of carbohydrates. In some embodiments, the carbohydrate source in the carrier material does not contain an anhydrous subunit. In some embodiments, the carbohydrate source in the carrier material includes a glycosidic linkage type distribution that is different from the glycosidic linkage type distribution of the oligosaccharide drug.

Accordingly, in some embodiments, a method of producing a nutritional composition includes combining an animal feed premix with a base nutritional composition.

V. Methods for providing animals with oligosaccharide preparations

In some embodiments, the methods described herein include providing oligosaccharide preparations to animals. In some embodiments, the animals are treated by feeding or providing an oligosaccharide drug. In some embodiments, the oligosaccharide drug is administered to animals at a specific targeted dose. The specific dose can be quantified, for example, as the mass of the oligosaccharide drug consumed by the animal

- 41 044359 per unit of time (for example, grams per day), or as the mass of the oligosaccharide preparation consumed by the animal per unit of time per unit of body weight of the animal (for example, mg of oligosaccharide per kg of body weight per day). In some embodiments, the specific dose of the oligosaccharide drug is 1, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170 , 180, 190, 200, 225, 250, 275, 300, 350, 400, 450, 500, 1000, 1500, 2000, 3000, 4000, 5000 or 10,000 mg/kg/day. In some embodiments, the weight of the oligosaccharide preparation is measured as the DP1+ content on a dry solids basis. In some embodiments, the weight of the oligosaccharide preparation is measured as the DP2+ content on a dry solids basis.

In some embodiments, the animals are provided with oligosaccharide preparations by oral administration in the form of nutritional compositions. In some embodiments, the nutritional composition includes an oligosaccharide preparation at a fixed concentration or inclusion level. The concentration or level of inclusion of the oligosaccharide in the nutritional composition can be quantified, for example, by the mass fraction of the oligosaccharide preparation per total weight of the final feed or nutritional composition. In some embodiments, the concentration or inclusion level is measured in parts per million (ppm) of the oligosaccharide on a solids basis for the final nutritional composition in its original state. In some embodiments, the concentration of the oligosaccharide drug is measured as the mass fraction of DP1+ particles on a dry solids basis. In some embodiments, the concentration of the oligosaccharide drug is measured as the mass fraction of DP2+ particles on a dry solids basis.

One of ordinary skill in the art is aware of various methods and techniques for determining the concentration of an oligosaccharide drug in a nutritional composition or final feed to achieve a given specific dose. For example, average daily feed intake by age has been established for various types of broiler chickens and can be used by a nutritionist or veterinarian to determine the appropriate level of inclusion in the final feed.

In some embodiments, oligosaccharide preparations are administered to animals by oral administration through ingested fluids. In some embodiments, the oligosaccharide preparations are provided in drinking water. In some embodiments, the concentration of the oligosaccharide drug in the drinking water is selected to provide the animal with a given, specific dose of the oligosaccharide drug.

VI. Selective modulation of the growth of gastrointestinal microorganisms

A. Selective modulation of the growth of beneficial gastrointestinal microorganisms

In some embodiments, the methods described herein include selectively enhancing or stimulating the growth of one or more species of microorganisms (e.g., bacteria) in the gastrointestinal tract of an animal, and in some embodiments, quantifying the level of one or more species of microorganisms (e.g., bacteria). In some embodiments, the composition of the animal's gastrointestinal microbiota is altered by the oligosaccharide drug toward a healthy state. In some embodiments, the microbial (eg, bacterial) species is beneficial to the animal (eg, beneficial to health).

In some embodiments, the microorganism species is an archaeal species. In some embodiments, the microorganism species is bacteria, viruses, bacteriophages, parasites, fungi, or protozoa. In some embodiments, the microorganism species is a bacterial species.

Bacteria disclosed herein include, but are not limited to, organisms classified in the genera Bacteroides, Odoribacter, Oscillibacter, Subdoligranulum, Biophila, Barnesiella or Ruminococcus. Examples of bacteria also include, but are not limited to, organisms classified in the genera Enterococcus, Lactobacillus, Propionibacterium, Bifidobacterium, or Streptococcus.

Microorganism species include, but are not limited to, Bacteroides clarus, Bacteroides dorei, Odoribacter splanchinicus and Barnesiella intestinihominis.

In some embodiments, the animal has a gastrointestinal microbiota comprising at least 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% , 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of at least one species of microorganism classified in the genera Bacteroides, Odoribacter, Oscillibacter, Subdoligranulum, Biophila, Barnesiella or Ruminococcus (e.g. when measured in a gastrointestinal sample as described herein). In some embodiments, the animal's gastrointestinal microbiota comprises at least 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of at least one species of microorganism classified as Enterococcus, Lactobacillus, Propionibacterium, Bifidobacterium or Streptococcus (for example, when measured in the gastrointestinal intestinal sample as described herein).

In some embodiments, the animal has a gastrointestinal microbiota comprising at least 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% , 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of at least one of Bacteroides clarus, Bacteroides dorei, Odoribacter

- 42 044359 splanchinicus or Barnesiella intestinihominis (for example, when measured in a gastrointestinal sample, as disclosed in this application).

In some embodiments, the animal has a gastrointestinal microbiota comprising at least 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% , 15%, 20%, 25%, 30%, 40% or 50% a combination of one or more species of microorganisms classified as the genera Bacteroides, Odoribacter, Oscillibacter, Subdoligranulum, Biophila, Barnesiella or Ruminococcus (for example, when measured in the gastrointestinal sample as described in this application). In some embodiments, the animal's gastrointestinal microbiota comprises at least 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, or 50% a combination of one or more species of microorganisms classified as the genera Enterococcus, Lactobacillus, Propionibacterium, Bifidobacterium, or Streptococcus (e.g., when measured in a gastrointestinal sample as described herein ).

In some embodiments, the animal has a gastrointestinal microbiota comprising at least 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% , 15%, 20%, 25%, 30%, 40%, or 50% a combination of one or more of Bacteroides clarus, Bacteroides dorei, Odoribacter splanchinicus, or Barnesiella intestinihominis (e.g., when measured in a gastrointestinal sample as described herein ).

B. Selective inhibition of the growth of pathogenic gastrointestinal microorganisms

In some embodiments, the methods described herein include selectively reducing or inhibiting the growth of one or more species of microorganisms (e.g., bacteria) in the gastrointestinal tract of an animal, and in some embodiments, quantifying the level of one or more species of microorganisms (e.g., bacteria). In some embodiments, the microbial (eg, bacterial) species is harmful to the animal (eg, harmful to the health of the animal). In some embodiments, the microbial (eg, bacterial) species is pathogenic to the animal. In some embodiments, the microbial (eg, bacterial) species is pathogenic to humans but not to animals. In some embodiments, the composition of the animal's gastrointestinal microbiota is altered by the oligosaccharide drug toward a healthy state.

In some embodiments, the microbial species is an archaeal species. In some embodiments, the microorganism species is a species of bacteria, virus, bacteriophage, parasite, fungus, or protozoan. In some embodiments, the microorganism species is a bacterial species.

Bacteria disclosed herein include, but are not limited to, bacteria of the phylum Proteobacteria. Bacteria also include, but are not limited to, organisms classified in the genera Helicobacter, Escherichia, Salmonella, Vibrio, or Yersinia. Examples of bacteria also include organisms classified in the genera Treponema, Streptococcus, Staphylococcus, Shigella, Rickettsia, Orientia, Pseudomonas, Neisseria, Mycoplasma, Mycobacterium, Listeria, Leptospira, Legionella, Emobsiochella, Eurolischinee, Enterocococcus, Coxiella, Corynebacterium, Clostridium, Chlamydia, Chlamydophila , Campylobacter, Burkholderia, Brucella, Borrelia, Bordetella, Bifidobacterium and Bacillus, but are not limited to. Microorganism species include, but are not limited to, Helicobacter pullorum, Proteobacteria johnsonii, Escherichia coli, Campylobacter jejuni and Lactobacillus crispatus. Microorganism species also include Aeromonas hydrophila, Campylobacter fetus, Plesiomonas shigelloides, Bacillus cereus, Campylobacter jejuni, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, enteroaggregative Escherichia coli, enterohemorrhagic Escherichia coli, enteroinvasive Escherichia coli, en terotoxigenic Escherichia coli, Helicobacter pylori, Klebsiellia pneumonia, Lysteria monocytogenes, Plesiomonas shigelloides, Salmonella spp., Salmonella typhi, Salmonella paratyphi, Shigella spp., Staphylococcus spp., Staphylococcus aureus, vancomycin-resistant Enterococcus spp., Vibrio spp., Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus and Yersinia enterocoli tica, but not limited to them. In some embodiments, the bacterium is resistant to one or more drugs.

In some embodiments, the animal has a gastrointestinal microbiota containing less than 50%, 40%, 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.1% of bacteria classified as Helicobacter, Proteobacteria, Escherichia, Campylobacter or Lactobacillus genera (eg, when measured in a gastrointestinal sample as disclosed herein). In some embodiments, the combination of bacteria classified as Helicobacter, Proteobacteria, Escherichia, Campylobacter, or Lactobacillus genera is less than 50%, 40%, 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6 %, 5%, 4%, 3%, 2%, 1% or 0.1% of the animal's gastrointestinal tract microbiota (eg, when measured in a gastrointestinal sample as described herein).

In some embodiments, the animal has a gastrointestinal microbiota containing less than 50%, 40%, 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.1% Helicobacter pullorum, Proteobacteria johnsonii, Escherichia coli, Campylobacter jejuni or Lactobacillus crispatus (eg, when measured in a gastrointestinal sample as described herein). In some embodiments, a combination of Helicobacter pullorum, Proteobacteria johnsonii, Escherichia coli,

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Campylobacter jejuni or Lactobacillus crispatus is less than 50%, 40%, 30%, 20%, 15%, 10%, 9%, 8%,

7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.1% of the animal's gastrointestinal tract microbiota (eg, when measured in a gastrointestinal sample as described herein).

C. Sampling and detection of gastrointestinal microorganisms

In some embodiments, the methods described herein include detecting or quantifying one or more species of microorganisms (eg, bacteria) in the gastrointestinal microbiota of an animal. In some embodiments, microbial (eg, bacterial) species are detected or quantified in a sample of the animal's gastrointestinal tract microbiota. Gastrointestinal microbiota samples can be obtained from an animal in any standard form that reflects the microbial content of the animal's gastrointestinal tract. Gastrointestinal microbiota samples include gastrointestinal tissue samples obtained, for example, by endoscopic biopsy. Gastrointestinal tissues include, for example, tissues of the oral cavity, esophagus, stomach, intestine, ileum, cecum, colon or rectum. Samples of feces, saliva and gastrointestinal ascitic fluid are also collected. Methods for obtaining gastrointestinal microbiota samples are standard and known to one skilled in the art.

In some embodiments, the sample is a single sample from a single animal. In some embodiments, the sample is a combination of multiple samples from a single animal. In some embodiments, microorganisms (eg, bacteria (eg, all bacteria)) are purified from the sample prior to analysis. In some embodiments, microorganisms (eg, bacteria) are purified from a single sample. In some embodiments, microorganisms (eg, bacteria) from multiple samples from a single animal are purified and subsequently pooled before analysis.

In some embodiments, total DNA or total RNA is isolated from the sample. Genomic DNA can be extracted from samples using standard methods known to one of ordinary skill in the art, including commercially available kits such as the Mo Bio Powersoil®-htp 96-well Soil DNA Extraction Kit (Mo Bio Laboratories, Carlsbad, CA), Mo Bio Powersoil® DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, CA) or QIAamp Stool DNA Mini Kit (QIAGEN, Valencia, CA) according to the manufacturer's instructions. RNA can be extracted from samples using standard assays known to those skilled in the art, including commercial kits such as the RNeasy PowerMicrobiome kit (QIAGEN, Valencia, Calif.) and the RiboPure Bacterial RNA Purification Kit (Life Technologies, Carlsbad, Calif.). Another method for isolating microbial (e.g., bacterial) RNA may involve enriching mRNA in purified bacterial RNA samples by removing tRNA. Alternatively, the RNA can be converted to cDNA which can be used to generate sequencing libraries using standard methods such as the Nextera XT sample preparation kit (Illumina, San Diego, CA).

Identification and determination of the relative abundance of microbial (e.g., bacterial) species in a sample can be accomplished by standard molecular biology techniques known to one of ordinary skill in the art, including, for example, genetic analysis (e.g., DNA sequencing (e.g., whole genome sequencing, whole genome sequencing shotgun (WSG)), RNA sequencing, PCR, quantitative PCR (qPCR), serology and antigen analysis, microscopy, metabolite identification, Gram staining, flow cytometry, immunological and culture-based methods such as colony-forming unit counts.

In some embodiments, the identification and relative abundance of microbial species (e.g., bacteria) is determined using whole genome shotgun sequencing (WGS), in which extracted DNA is fragmented into pieces of varying lengths (from 300 to about 40,000 nucleotides) and directly sequenced without amplification. Sequence data may be obtained using any sequencing technology, including, for example, but not limited to, Sanger, Illumina, 454 Life Sciences, Ion Torrent, ABI, Pacific Biosciences, and/or Oxford Nanopore.

Sequencing libraries for microbial (eg, bacterial) whole genome sequencing (WGS) can be generated from microbial (eg, bacterial) genomic DNA. For genomic DNA isolated from an animal sample, the DNA can be enriched with microbial (eg, bacterial) DNA if necessary using commercial kits such as the NEBNext Microbiome DNA Enrichment Kit (New England Biolabs, Ipswich, MA) or other enrichment kit. Sequencing libraries can be prepared from genomic DNA using also commercial kits such as the Nextera Mate-Pair Sample Preparation Kit, TruSeq DNA PCR-Free or TruSeq Nano DNA, or the Nextera XT Sample Preparation Kit (Illumina, San Francisco). Diego, California) according to manufacturer's instructions.

Alternatively, libraries can be produced using other compatible kits

- 44 044359 with an Illumina sequencing platform, such as the NEBNext DNA Library Construction Kit (New England Biolabs, Ipswich, MA). The libraries can then be sequenced using standard sequencing technology, including but not limited to a MiSeq, HiSeq or

NextSeq (Illumina, San Diego, CA).

Alternatively, a whole genome shotgun sequencing fragment library generated using standard methods in the art may be used. For example, a library of fragments from shotgun sequencing can be generated using the GS FLX Titanium Rapid Library Preparation Kit (454 Life Sciences, Branford, CT), amplified using the GS FLX Titanium emPCR kit (454 Life Sciences, Branford, CT), and sequenced according to standard 454 pyrosequencing protocols on a 454 sequencer (454 Life Sciences, Branford, CT).

Nucleic acid sequences can be analyzed to determine taxonomic assignments using sequence similarity and phylogenetic placement methods or a combination of these two strategies. A similar approach can be used to annotate protein names, protein functions, transcription factor names, and any other nucleic acid sequence classification scheme. Methods based on sequence similarity include BLAST, BLASTx, tBLASTn, tBLASTx, RDP-classifier, DNAclust, RapSearch2, DIAMOND, USEARCH and various implementations of these algorithms such as QIIME or Mothur. These methods match the read sequence to a reference database and select the best match. Common databases include KEGG, MetaCyc, NCBI non-redundant database, Greengenes, RDP and Silva for taxonomic assignments. For functional assignment, reads are mapped to various functional databases such as COG, KEGG, BioCyc, MetaCyc and the Carbohydrate Active Enzyme (CAZy) database. Classes of microorganisms are assigned using software including MetaPhlAn.

In some embodiments, bacterial constituents are identified by characterizing the DNA sequence of the bacterial 16S small subunit ribosomal RNA gene (16S rRNA gene). The 16S rRNA gene is approximately 1500 nucleotides long and is generally highly conserved across all organisms, but contains specific variable and hypervariable regions (V1-V9) that have sufficient nucleotide diversity to differentiate taxa at the species and strain level in most organisms. These regions in bacteria are defined by nucleotides 69-99, 137-242, 433-497, 576-682, 822-879, 986-1043, 1117-1173, 1243-1294 and 1435-1465, respectively, using a numbering system based on E. coli nomenclature.

The composition of a bacterial community can be determined by sequencing the entire 16S rRNA gene or at least one of the V1, V2, V3, V4, V5, V6, V7, V8, and V9 regions of that gene, or by sequencing any combination of variable regions of that gene (e.g. , V1-3 or V3-5). In one embodiment, regions V1, V2 and V3 are used to characterize the microbiota. In another embodiment, the V3, V4 and V5 regions are used to characterize the microbiota. In another embodiment, the V4 region is used to characterize the microbiota.

Sequences that are at least 97% identical to each other are grouped into operational taxonomic units (OTUs). OTUs that contain sequence with 97% similarity roughly correspond to species-level taxa. At least one representative sequence from each OTU is selected and used to obtain a taxonomic assignment for the OTU by comparison to a reference database of curated 16S rRNA gene sequences (eg, Greengenes or SILVA databases). The relationships among OTUs in a microbial community can be inferred by constructing a phylogenetic tree from the representative sequences of each OTU. Using known methods to determine the complete 16S sequence or the sequence of any variable region of the 16S sequence, genomic DNA is extracted from the bacterial sample, 16S rRNA (the complete region or specific variable regions) is amplified using polymerase chain reaction (PCR), the PCR products are purified, and the nucleotide sequences are delineated to determine the genetic composition of the 16S rRNA gene or variable region of the gene. If full 16S sequencing is performed, Sanger sequencing may be used, but is not limited to. If one or more variable regions are used, such as the V4 region, sequencing can be performed using the Sanger method or a next generation sequencing method such as, but not limited to, the Illumina method. Primers designed to anneal to conserved regions of the 16S rRNA genes (e.g., primers 515F and 805R to amplify the V4 region) can contain unique barcode sequences that allow the simultaneous characterization of multiple microbial communities.

In addition to the 16S rRNA gene, to assess microbial community composition, a selected set of genes known to be marker genes for a given species or taxonomic group are analyzed. Alternatively, these genes are analyzed using a PCR-based screening strategy. For example, different strains of pathogenic Escherichia coli are distinguished by genes encoding

- 45 044359 heat-labile (LTI, LTIIa and LTIIb) and heat-stable (STI and STII) toxins, verotoxins types 1, 2 and 2e (VT1, VT2, and VT2e, respectively), cytotoxic necrotic factors (CNF1 and CNF2), attachment mechanisms and displacement (eaeA), enteroaggregative mechanisms (Eagg) and enteroinvasive mechanisms (Einv). The optimal genes to use for determining the taxonomic composition of a microbial community using marker genes will be familiar to one of ordinary skill in the art of sequence-based taxonomic identification.

In some embodiments, the identity of the microbial composition is characterized by the identification of nucleotide markers or genes, particularly highly conserved genes (eg, housekeeping genes) or combinations thereof. Using certain methods, DNA isolated from a bacterial sample will have specific regions of the genome amplified by PCR and sequenced to determine the nucleotide sequence of the amplified products.

D. Transcription of microbial genes

In some embodiments, the methods described herein include stimulating the expression of one or more microbial (eg, bacterial) proteins in the gastrointestinal tract of an animal. In some embodiments, the microbial protein is a bacterial protein. In some embodiments, the microbial (eg, bacterial) protein is a hydrolytic enzyme, a protein involved in digestion (eg, hydrolytic enzymatic digestion), or a protein involved in metabolism. Microbial proteins include carbohydrate-active enzyme (CAZymes), polysaccharide utilization loci (PUL) sus-like protein, GH127 (CAZyme a-L-arabinofuranosidase), susC (starch-binding outer membrane protein) and susD (starch-binding protein), but are not limited to them.

Microbial (eg, bacterial) proteins described herein can be detected and quantified using standard molecular biology techniques. For example, the level of microbial protein expression can be determined in a gastrointestinal sample of an animal using RNA (eg, RNA sequencing, shotgun sequencing, quantitative PCR) or protein expression (eg, ELISA, Western blotting, other immunological methods).

VII. Ways to increase animal productivity

A. Feed Conversion Ratio

In some embodiments, the methods described herein include reducing the animal's feed conversion ratio. In some embodiments, an animal fed a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition as described herein has a lower feed conversion ratio compared to an animal fed a diet that does not include the synthetic oligosaccharide. a drug. As used herein, the term feed conversion ratio (FCR) refers to the ratio of the input mass of feed (eg, consumed by an animal) to the animal product, the animal product being the target animal product. For example, the animal product for dairy animals is milk, and the animal product for animals raised for meat is body weight.

In some embodiments, the animal is raised for meat and the target animal product is body weight. Thus, in some embodiments, FCR refers to the ratio of the weight of feed consumed to the final body weight of the animal before processing. In some embodiments, FCR refers to the ratio of the weight of feed consumed to the final weight gain of the animal before processing. It should be understood that FCR can be measured for an animal or population of animals over different time periods. For example, in some embodiments, the FCR is an FCR throughout the life of the animal. In other embodiments, the FCR is a daily FCR, or a weekly FCR, or a cumulative FCR measured up to a specific point in time (eg, a specific day).

One skilled in the art will appreciate that the target minimum FCR (optimal FCR) may be different for different types of animals and can be different for different breeds of the same type of animal (eg, different breeds of broiler chickens, or different breeds of pigs). The minimum FCR of target production may also vary depending on the age of the animal (eg, chicks or pigs in the first finishing period versus the second finishing period) or the sex of the animal. It should be clear that the optimal FCR may differ depending on any combination of these factors.

The minimum target usually refers to the lowest nutritional efficiency observed for a given animal and breed under ideal rearing conditions, ideal animal health and ideal dietary nutrition. Those skilled in the art are well aware that under normal rearing conditions an animal may not achieve the minimum target FCR. An animal may not achieve its minimum target FCR due to various factors related to health, nutrition, environment and/or community. The animal may not achieve its goal

- 46 044359 minimum planned FCR when grown in a challenging environment, which may include, for example, pathogenic environmental stress, excessive environmental temperature (heat stress), excessive environmental humidity, crowding or other social interaction effects such as difficulty accessing feed or drinking water. In some embodiments, the animal may not achieve its minimum target FCR due to disease or pathogenic environmental stress. In other embodiments, the animal may not achieve its minimum target FCR due to excessive environmental temperature (heat stress) or excessive environmental humidity. In other embodiments, the animal may not achieve its minimum target FCR due to crowding or other social interaction effects, such as difficulty accessing food or drinking water.

In some embodiments, an animal fed a diet not including a synthetic oligosaccharide drug described herein has an FCR that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8% %, 9% or 10% higher than the minimum planned FCR. In some embodiments, an animal fed a diet that does not include a synthetic oligosaccharide drug described herein has an FCR that is 1-10% above the minimum target, 2-10% above the minimum target, or 5-10% above the minimum planned indicator.

In some embodiments, an animal receiving a nutritional composition comprising a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition as described herein has an FCR that is closer to the minimum target compared to an animal receiving a diet that does not include a synthetic oligosaccharide drug. In specific embodiments, an animal fed a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition as described herein has an FCR that is 0-10% higher than the minimum target, 0-5% higher the minimum planned indicator or 0-2% above the minimum planned indicator.

In some embodiments, an animal fed a synthetic oligosaccharide drug, nutritional composition, animal feed premix, or animal feed composition as described herein has a lower feed conversion ratio compared to an animal fed a diet that does not include the synthetic oligosaccharide drug . For example, in some embodiments, an animal fed a diet including a synthetic oligosaccharide drug consumes less feed but produces the same animal product as an animal fed a diet not including the synthetic oligosaccharide drug. In other embodiments, an animal fed a diet including a synthetic oligosaccharide drug consumes the same amount of feed but produces a higher animal product compared to an animal fed a diet not including the synthetic oligosaccharide drug. In other embodiments, an animal fed a diet including a synthetic oligosaccharide drug consumes less food and produces a higher animal product compared to an animal fed a diet not including the synthetic oligosaccharide drug.

In some embodiments, the FCR of an animal fed a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition as described herein is reduced by at least 1%, by at least 2%, by at least 4%, at least 6%, at least 8%, at least 10%, at least 12%, 1-10%, 4-10%, 1-8%, at 4-8%, 1-6% or 4-6% compared to an animal fed a diet that did not include a synthetic oligosaccharide drug. In some embodiments, the animal is a poultry. In some embodiments, the poultry FCR is reduced at 0 to 14 days of age, 15 to 28 days, 29 to 35 days, greater than 35 days, greater than 42 days, greater than 6 weeks, greater than 6.5 weeks, 0 to 35 days, 0 to 42 days, 0 to 6 weeks, 0 to 6.5 weeks, 15 to 35 days, 36 to 42 days, 15 to 39 days, or 40 to 46 days.

In one embodiment, the 35 day FCR for poultry fed a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition as described herein is reduced by 4-6% compared to poultry fed the diet , which does not include a synthetic oligosaccharide drug. For example, in a certain embodiment, the FCR per 35 days for poultry fed a nutritional composition containing a synthetic oligosaccharide drug, nutritional composition, animal feed premix, or animal feed composition as described herein is 1.53, FCR per 35 days for poultry fed a diet without a synthetic oligosaccharide drug is 1.61, and the FCR of birds fed a nutritional composition containing an oligosaccharide drug, nutritional composition, animal feed premix or animal feed composition is reduced by approximately 5% compared to birds fed a diet without a synthetic oligosaccharide drug. In some embodiments, an FCR of 42 days, greater than 6 weeks, or greater than 6.5 weeks for poultry fed the synthetic oligosaccharide

- 47 044359 drug, nutritional composition, animal feed premix or animal feed composition, as described in this application, is reduced by 4-6% compared to poultry fed a diet that does not include a synthetic oligosaccharide drug.

In some embodiments, a population of animals fed a synthetic oligosaccharide drug, nutritional product, animal feed premix, or animal feed composition as described herein has a lower FCR compared to a population of animals fed a diet that does not include the synthetic oligosaccharide drug. where FCR is adjusted for mortality in the animal population.

In some embodiments, an animal fed a synthetic oligosaccharide drug, animal feed premix, or animal feed composition has a lower FCR than an animal fed a diet that does not include the synthetic oligosaccharide drug but includes one or more antibiotics, one or more ionophores, soluble corn fiber, modified wheat starch or yeast mannan, or any combination thereof.

Those skilled in the art will know that when determining FCR, it can be adjusted for mortality to reduce errors due to small statistical data. Methods for adjusting FCR for mortality are well known to those skilled in the art.

In some embodiments, which may be combined with any of the above embodiments, poultry is a single poultry bird, while in other embodiments, poultry is a population of poultry birds.

In some embodiments, the animal is poultry and the animal feed composition is a poultry feed, wherein the synthetic oligosaccharide drug, poultry nutritional composition, poultry feed premix, or poultry feed composition reduces the feed conversion ratio (FCR). ) by about 10%, or by about 5%, or by 1-10%, by 2-10%, by 3-10%, by 4-10%, by 5-10%, by 2-5%, by 2-6%, by 2-7%, by 2-8%, by 2-9% or by 1-5% when fed to poultry, compared to poultry receiving a feed composition without a synthetic oligosaccharide preparation.

In some embodiments, the poultry suffers from a disease or disorder, or is raised in an unfavorable environment, where the synthetic oligosaccharide drug, poultry nutritional composition, poultry feed premix, or poultry feed composition reduces the feed conversion ratio (FCR) by approximately 30% , about 25%, about 20%, about 15%, about 10% or about 5%, or from 1 to 30%, from 5 to 30%, from 10 to 30%, from 5 to 20% , from 10 to 20%, from 1 to 20%, from 1 to 15%, from 1 to 10%, from 2 to 10%, from 3 to 10%, from 4 to 10%, from 5 to 10%, from 2 to 5%, 2 to 6%, 2 to 7%, 2 to 8%, 2 to 9% or 1 to 5% when fed to poultry compared to poultry fed a feed composition without synthetic oligosaccharide drug.

In some embodiments, the animal is a pig and the animal feed composition is a swine feed, wherein the synthetic oligosaccharide drug, swine nutritional composition, swine feed premix, or swine feed composition reduces the feed conversion ratio (FCR) by about 15% , by about 10% or by about 5%, or from 1 to 15%, from 2 to 15%, from 3 to 15%, from 4 to 15%, from 5 to 15%, from 10 to 15%, from 1 up to 10%, from 2 to 10%, from 3 to 10%, from 4 to 10%, from 5 to 10%, from 2 to 5%, from 2 to 6%, from 2 to 7%, from 2 to 8 %, from 2 to 9% or from 1 to 5% when fed to pigs, compared to pigs receiving a feed composition without a synthetic oligosaccharide preparation.

In some embodiments, the pig suffers from a disease or disorder, or is raised in an unfavorable environment, where the synthetic oligosaccharide drug, swine nutritional composition, swine feed premix, or swine feed composition reduces the feed conversion ratio (FCR) by about 40%, by about 35 %, about 30%, about 25%, about 20%, about 15%, about 10% or about 5%, or from 1 to 40%, from 5 to 40%, from 10 to 40% , from 15 to 40%, from 20 to 40%, from 25 to 40%, from 30 to 40%, from 1 to 30%, from 5 to 30%, from 10 to 30%, from 5 to 20%, from 10 to 20%, from 1 to 20%, from 1 to 15%, from 1 to 10%, from 2 to 10%, from 3 to 10%, from 4 to 10%, from 5 to 10%, from 2 to 5%, 2 to 6%, 2 to 7%, 2 to 8%, 2 to 9%, or 1 to 5% when fed to pigs, compared to pigs receiving a feed composition without a synthetic oligosaccharide preparation.

B. Body weight

In some embodiments, an animal in accordance with the invention that receives a synthetic oligosaccharide drug, nutritional composition, animal feed premix, or animal feed composition described herein may exhibit an increase in body weight compared to a control animal not receiving the oligosaccharide drug. a nutritional composition, an animal feed premix or an animal feed composition. In some embodiments, both the animal of the invention and the control animal consume the same amount of food based on body weight, but the animal of the invention fed the synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or feed composition for animals, shows an increase in weight gain compared to the control animal that received a diet that did not include a synthetic oligosaccharide drug.

An animal's weight gain can be determined by any suitable methods known in the art. For example, to determine the weight gain of an animal that is subjected to a feeding regimen of a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition, one skilled in the art may measure the animal's body weight before the feeding regimen, measure the animal's weight after the animal will receive a synthetic oligosaccharide preparation, nutritional composition, animal feed premix or animal feed composition, and determine the difference between these two measurements.

In some embodiments, the gain may be average daily weight gain (also called average daily weight gain (ADG)), average weekly weight gain (AWG), or total weight gain (BWG).

C. Average daily weight gain

In some embodiments, providing an animal with a synthetic oligosaccharide drug, nutritional composition, animal feed premix, or animal feed composition results in increased average daily weight gain compared to an animal fed the diet without the synthetic oligosaccharide drug. In some embodiments, providing a population of animals with a synthetic oligosaccharide drug, nutritional composition, animal feed premix, or feed composition results in an increase in average daily weight gain, compared to a group of animals receiving the diet without the synthetic oligosaccharide drug.

In one embodiment, an animal's average daily weight gain is the weight gained each day by an individual animal, averaged over a given period of time. In some embodiments, the average daily gain for a population of animals is the average daily gain for each individual animal averaged over the population; where average daily weight gain is the weight gained each day by an individual animal, averaged over a given period of time. In other embodiments, the average daily weight gain for a population of animals is the total weight gained by the population each day, divided by the number of individual animals in the population, averaged over a given period of time. It should be understood that daily gain or average daily gain can be further averaged, for example, to provide average daily gain among animal populations.

In some embodiments, the animal is a poultry and the poultry receiving the synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition has an average daily weight gain of at least 20 g per day, at least 30 g per day , at least 40 g per day, at least 50 g per day, at least 60 g per day, at least 70 g per day, at least 80 g per day, at least 90 g per day, from 20 to 100 g per day, 20 to 80 g per day, 30 to 50 g per day, 40 to 60 g per day, 50 to 70 g per day, or 70 to 90 g per day. In one embodiment, the animal is a poultry, and the poultry receiving the synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition has an average daily weight gain of at least 50 grams per day. In some embodiments, poultry fed the synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition has an average daily gain of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 9%, at least 10%, at least 11%, at least at least 12%, 1-10%, 2-8%, or 3-5% more than the average daily weight gain of poultry fed a diet that does not include a synthetic oligosaccharide drug.

In some embodiments, the animal is a poultry, and the poultry is between 0 and 14 days old and the average daily weight gain is at least 30 g, at least 40 g, or at least 50 g per day.

In other embodiments, the animal is a poultry, the poultry is between 14 and 28 days old, and the average daily weight gain is at least 70 g, at least 80 g, or at least 90 g per day.

In yet other embodiments, the animal is a poultry, the poultry is between 29 and 35 days old, and the average daily weight gain is at least 50 g, at least 60 g, or at least 70 g per day.

In some embodiments, which may be combined with the foregoing, the animal is a poultry and the animal feed composition is a poultry feed, wherein the synthetic oligosaccharide preparation, the poultry nutritional composition, the poultry feed premix or a poultry feed composition increases the bird's average daily growth rate to about 10%, or about 5%, or 1 to 10%, 2 to 10%, 3 to 10%, 4 to 10%, 5 to 10% , from 2 to 5%, from 2 to 6%, from 2 to 7%, from 2 to 8%, from 2 to 9% or from 1 to 5% when fed to poultry, compared to poultry receiving the feed composition without synthetic oligosaccharide drug.

In some embodiments, the poultry is suffering from a disease or disorder or is being raised in an unfavorable environment where the synthetic oligosaccharide preparation, poultry nutritional composition, poultry feed premix, or poultry feed composition increases the average daily weight gain of the poultry by about 30%, by about 30%. 25%, about 20%, about 15%, about 10% or about 5%, or from 1 to 30%, from 5 to 30%, from 10 to 30%, from 5 to 20%, from 10 up to 20%, from 1 to 20%, from 1 to 15%, from 1 to 10%, from 2 to 10%, from 3 to 10%, from 4 to 10%, from 5 to 10%, from 2 to 5 %, from 2 to 6%, from 2 to 7%, from 2 to 8%, from 2 to 9% or from 1 to 5% when fed to poultry, compared to poultry receiving a feed composition without a synthetic oligosaccharide preparation.

In some embodiments, which may be combined with the foregoing, the animal is a pig and the animal feed composition is a pig feed, wherein the synthetic oligosaccharide preparation, swine nutritional preparation, swine feed premix, or swine feed composition increases average daily weight gain pigs by about 15%, by about 10% or by about 5%, or from 1 to 15%, from 2 to 15%, from 3 to 15%, from 4 to 15%, from 5 to 15%, from 10 to 15%, from 1 to 10%, from 2 to 10%, from 3 to 10%, from 4 to 10%, from 5 to 10%, from 2 to 5%, from 2 to 6%, from 2 to 7% , from 2 to 8%, from 2 to 9% or from 1 to 5% when fed to pigs compared to pigs receiving a feed composition without a synthetic oligosaccharide preparation.

In some embodiments, the pig suffers from a disease or disorder, or is raised in an unfavorable environment, wherein the oligosaccharide preparation, swine nutritional composition, swine feed premix, or swine feed composition increases the pig's average daily growth rate by about 40%, by about 35%, by about 30%, about 25%, about 20%, about 15%, about 10% or about 5%, or from 1 to 40%, from 5 to 40%, from 10 to 40%, from 15 to 40%, from 20 to 40%, from 25 to 40%, from 30 to 40%, from 1 to 30%, from 5 to 30%, from 10 to 30%, from 5 to 20%, from 10 to 20% , from 1 to 20%, from 1 to 15%, from 1 to 10%, from 2 to 10%, from 3 and 10%, from 4 to 10%, from 5 to 10%, from 2 to 5%, from 2 to 6%, 2 to 7%, 2 to 8%, 2 to 9%, or 1 to 5%, when fed to pigs, compared to pigs receiving a feed composition without a synthetic oligosaccharide preparation.

In some embodiments, the animal is a pig and the pig fed the synthetic oligosaccharide preparation, swine nutritional preparation, swine feed premix, or swine feed composition has an average daily weight gain of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 9%, at least 10%, at least at least 11%, at least 12%, 1 to 10%, 2 to 8%, or 3 to 5% more than the average daily weight gain of pigs fed a diet that does not include a synthetic oligosaccharide preparation.

D. Average weekly gain

In some embodiments, providing an animal with a synthetic oligosaccharide drug, nutritional composition, animal feed premix, or animal feed composition results in an increase in average weekly weight gain compared to an animal fed a diet without the synthetic oligosaccharide drug. In some embodiments, providing a population of animals with a synthetic oligosaccharide drug, nutritional composition, animal feed premix, or feed composition results in increased average weekly weight gain compared to a population of animals fed the diet without the synthetic oligosaccharide drug.

In one embodiment, the average weekly weight gain of an animal is the weight gained each week by an individual animal, averaged over a given period of time. In some embodiments, the average weekly weight gain for a population of animals is the average weekly weight gain for each individual animal averaged over the population; where average weekly weight gain is the weight gained each week by an individual animal, averaged over a given period of time. In other embodiments, the average weekly weight gain for a population of animals is the total weight gained by the population each week divided by the number of individual animals in the population averaged over a given period of time. It should be understood that the average weekly gain may be further averaged, for example, to provide an average weekly gain across animal populations.

In some embodiments, the animal is a poultry, and the poultry

- 50 044359 poultry receiving a synthetic oligosaccharide preparation, nutritional composition, animal feed premix or animal feed composition has an average weekly gain of at least 100 g per week, at least 200 g per week, at least 300 g per week , at least 400 g per week, at least 500 g per week, at least 600 g per week, at least 700 g per week, at least 800 g per week, from 100 to 800 g per week, from 100 to 400 g per week, 300 to 600 g per week, 500 to 800 g per week, or 350 to 550 g per week. In one embodiment, poultry fed with the synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition have an average weekly gain of at least 400 g per week. In some embodiments, poultry fed the synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition have an average weekly gain of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 9%, at least 10%, at least 11%, at least at least 12%, 1-10%, 2-8% or 3-5% more than the average weekly gain of poultry fed a diet that does not include an oligosaccharide drug.

In some embodiments, the animal is a pig and the pig fed the synthetic oligosaccharide preparation, swine nutritional composition, swine feed premix, or swine feed composition has an average weekly gain of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 9%, at least 10%, at least at least 11%, at least 12%, 1-10%, 2-8% or 3-5% more than the average weekly weight gain of pigs fed a diet that does not include a synthetic oligosaccharide drug.

E. Final weight gain

In some embodiments, providing an animal with a synthetic oligosaccharide drug, nutritional composition, animal feed premix, or animal feed composition results in an increase in final weight gain, compared to an animal receiving the diet without the synthetic oligosaccharide drug. In some embodiments, providing a population of animals with a synthetic oligosaccharide drug, nutritional composition, animal feed premix, or feed composition results in an increase in average net weight gain compared to a population of animals fed the diet without the synthetic oligosaccharide drug.

In some embodiments, providing an animal or population of animals with a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition results in a final weight gain or average final weight gain that is closer to the maximum performance targets than the animals or animal population receiving food without synthetic oligosaccharide preparation. The maximum performance target usually refers to the highest practical gain observed for a given animal type and breed under ideal rearing conditions, ideal animal health and ideal dietary nutrition.

In one embodiment, the final weight gain is the amount of weight gained by an individual animal over a period of time. For example, in one embodiment, total weight gain is the amount of weight that an individual animal gains from 0 days of age to the last weight taken before the animal was processed, or the final weight taken on the day the animal was processed. For example, in one embodiment, the total weight gain from days 0 to 28 for an animal is the amount of weight that an individual animal gains from 0 days of age to 28 days of age.

In another embodiment, average total weight gain is the amount of weight that an individual animal gains over a period of time, averaged over a population of animals. For example, in one embodiment, the average total weight gain is the amount of weight that an individual animal gains from 0 days of age to the final weight taken before the animal was processed, or the final weight taken on the day the animal was processed, the animal averaged over the animal population. In yet another embodiment, average total weight gain is the amount of weight that a population of animals gains over a period of time divided by the number of individual animals in the population. For example, in one embodiment, the average total weight gain is the amount of weight gained by a population of animals from 0 days of age to the final weight taken before the animal population was processed, or the final weight taken on the day the animal was processed, divided by the number of individual animals in the population.

It should be understood that the total weight gain and average total weight gain may be further averaged. For example, the average weight gain for different populations of the same animal type can be averaged to obtain the average overall weight gain for the populations.

In some embodiments, the animal is poultry, and the poultry treated with the synthetic oligosaccharide drug, nutritional composition, premix

- 51 044359 animal feed or feed composition for animals, has a final weight gain of at least 3 kg, at least 2.5 kg, at least 2 kg, at least 1.5 kg, at least 1 kg, from 1 to 3 kg or from 1.5 to 2.5 kg. In one embodiment, poultry fed the synthetic oligosaccharide preparation, animal feed premix, or animal feed composition have a final weight gain of at least 2 kg. In some embodiments, poultry fed the synthetic oligosaccharide preparation, animal feed premix, or animal feed composition have a final weight gain of at least 1%, at least 2%, at least 3%, at least by 4%, by at least 5%, by at least 6%, by at least 8%, by at least 9%, by at least 10%, by at least 11%, by at least 12%, 1-10%, 2-8% or 3-5% more than the final weight gain of birds fed a diet that did not include a synthetic oligosaccharide drug. In some embodiments, poultry fed the synthetic oligosaccharide preparation, animal feed premix, or animal feed composition have a final weight gain of at least 0.01 kg, at least 0.02 kg, or at least 0.03 kg, at least 0.04 kg, at least 0.05 kg, at least 0.06 kg, at least 0.07 kg, at least 0.08 kg, at least 0.09 kg, at least 0.1 kg, 0.01-0.1 kg, 0.03-0.07 kg or 0.04-0.06 kg more than the final weight gain of the bird , who received a diet that did not include a synthetic oligosaccharide drug.

In some embodiments, the animal is a poultry and the poultry receiving the synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition has an average final weight gain of at least 3 kg, at least 2.5 kg, at least 2 kg, at least 1.5 kg, at least 1 kg, from 1 to 3 kg or from 1.5 to 2.5 kg. In one embodiment, poultry fed the synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition have an average final weight gain of at least 2 kg. In some embodiments, poultry fed the synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition have an average final weight gain of at least 1%, at least 2%, at least 3%, by at least 4%, by at least 5%, by at least 6%, by at least 8%, by at least 9%, by at least 10%, by at least 11%, according to at least 12%, 1-10%, 2-8%, or 3-5% more than the average final weight gain of poultry fed a diet that does not include a synthetic oligosaccharide preparation. In some embodiments, poultry fed the synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition have an average final weight gain of at least 0.01 kg, at least 0.02 kg, at least by 0.03 kg, by at least 0.04 kg, by at least 0.05 kg, by at least 0.06 kg, by at least 0.07 kg, by at least 0.08 kg , at least 0.09 kg, at least 0.1 kg, 0.01-0.1 kg, 0.03-0.07 kg or 0.04-0.06 kg more than average the final weight gain of birds fed a diet that did not include a synthetic oligosaccharide drug.

In some embodiments, the animal is a poultry, and the poultry is between 0 and 14 days, 15 and 28 days, 29 and 35 days, 0 and 42 days, 0 and 6 weeks, or 0 and 6.5 weeks. In some embodiments, the start phase is from 0 to 14 days, the growth phase is from 15 to 28 days, and the finishing phase is from 29 to 35 days. In other embodiments, the starting phase is from 0 to 14 days, the growing phase is from 15 to 35 days, and the finishing phase is from 36 to 42 days. In other embodiments, the start phase is from 0 to 14 days, the growth phase is from 15 to 39 days, and the finishing phase is from 40 to 46 days. It should be understood that the duration of the starter phase, rearing phase and finishing phase for poultry may vary depending on the intended use of the poultry or poultry product. For example, in some embodiments, the duration of the starter phase, grower phase, and finisher phase may be different if the poultry is destined for broiler chicken production versus processing for tray-packed chicken meat.

In some embodiments, which may be combined with any of the above embodiments, poultry is a single poultry bird, while in other embodiments, poultry is a population of poultry birds.

In some embodiments, a pig fed the synthetic oligosaccharide preparation, swine nutritional composition, swine feed premix, or swine feed composition has a final weight gain of at least 1%, at least 2%, at least 3%, by at least 4%, by at least 5%, by at least 6%, by at least 8%, by at least 9%, by at least 10%, by at least 11%, according to at least 12%, 1-10%, 2-8% or 3-5% more than the final weight gain of pigs fed a diet that does not include a synthetic oligosaccharide drug.

- 52 044359

In some embodiments, a pig fed the synthetic oligosaccharide preparation, swine nutritional composition, swine feed premix, or swine feed composition has an average final weight gain of at least 1%, at least 2%, or at least 3% , at least 4%, at least 5%, at least 6%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, 1-10%, 2-8%, or 3-5% more than the average final weight gain of pigs fed a diet that does not include a synthetic oligosaccharide preparation.

In some embodiments, which may be combined with any of the above embodiments, the pig is an individual pig, while in other embodiments, the pig is a population of pigs.

F. Animal Product Yield

In some embodiments, providing an animal with a synthetic oligosaccharide drug, nutritional composition, animal feed premix, or animal feed composition as described herein results in increased yield of the animal product compared to animals fed a diet that does not include the synthetic oligosaccharide drug. In some embodiments, an animal receiving a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition provides at least 1%, at least 2%, at least 3%, at least an increase in 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, 1-10% , 4-10%, 6-10% or 2-8% more animal product, compared to an animal fed a diet that does not include a synthetic oligosaccharide drug. For example, in some embodiments, the animal product is the meat of an animal, and an animal treated with a synthetic oligosaccharide drug as described herein produces a greater amount of meat compared to an animal not treated with the oligosaccharide drug. In some embodiments, providing an animal population with a synthetic oligosaccharide drug, nutritional composition, animal feed premix, or animal feed composition results in an increase in the average yield of the animal product compared to an animal population fed a feed that does not include the synthetic oligosaccharide drug. In some embodiments, the average animal product yield is the amount of animal product obtained from each individual animal averaged over a population of animals.

In some embodiments, the animal product is meat from an animal (eg, which may be sold to consumers, processed into a food product, or consumed by humans). In some embodiments, the animal is poultry and the animal product is a poultry eviscerated carcass, poultry eviscerated leg meat, poultry eviscerated breast meat, poultry eviscerated drumstick meat, poultry eviscerated fat. , breast fillet from a poultry carcass or chicken leg fillet from a poultry carcass. In other embodiments, the animal is poultry and the animal product is white meat, breast fillet, and breast meat. In another embodiment, the animal is poultry and the product is tray-packaged chicken. In yet another embodiment, the animal is poultry and the product is a whole bird without giblets (WOG).

In some embodiments, the yield of the animal product is the yield of the product from an individual animal. In some embodiments, the average yield of an animal product is the yield obtained from each individual animal in a population of animals, averaged over the population. In yet another embodiment, the average animal product yield is the total animal product yield obtained from a population of animals divided by the number of individual animals in the animal population.

In some embodiments, the animal is poultry, the leg meat yield from the eviscerated poultry carcass is at least 6%, at least 8%, at least 10%, at least 12%, 6 to 12%, 8 up to 12%, 10 to 18%, 12 to 16% or 12 to 14% of the live weight of poultry fed with a synthetic oligosaccharide preparation, nutritional composition, animal feed premix or animal feed composition. In some embodiments, the yield of leg meat from an eviscerated poultry carcass treated with a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition as described herein is at least 1%, at least 2% %, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 9%, at least 10% , at least 11%, at least 12%, 1-10%, 2-8% or 3-5% more than poultry fed a diet that does not include a synthetic oligosaccharide preparation.

In some embodiments, the animal is a domestic bird

- 53 044359 tsu, and the average yield of leg meat from a gutted poultry carcass is at least 6%, at least 8%, at least 10%, at least 12%, from 6 to 12%, from 8 to 12% , from 10 to 18%, from 12 to 16% or from 12 to 14% of the live weight of poultry fed with a synthetic oligosaccharide preparation, nutritional composition, animal feed premix or animal feed composition. In some embodiments, the average yield of leg meat from an eviscerated poultry carcass fed a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition as described herein is at least 1%, at least 2% %, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 9%, at least 10% , at least 11%, at least 12%, 1-10%, 2-8% or 3-5% more than poultry fed a diet that does not include a synthetic oligosaccharide preparation.

In some embodiments, the animal is poultry and the breast meat yield from the poultry is at least 10%, at least 12%, at least 15%, at least 16%, at least 18%, at least 20%, at least 22%, at least 24%, at least 28%, 10 to 18%, 12 to 16%, 18 to 29%, 20 to 27% or 20 up to 25% of the live weight of birds receiving a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition. In some embodiments, the yield of breast meat from an eviscerated poultry carcass treated with a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition as described herein is at least 1%, at least 2% %, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 9%, at least 10% , at least 11%, at least 12%, 1-10%, 2-8% or 35% more than poultry fed a diet that does not include a synthetic oligosaccharide preparation.

In some embodiments, the animal is poultry and the average breast meat yield from a gutted poultry carcass is at least 10%, at least 12%, at least 15%, at least 16%, at least 18% , at least 20%, at least 22%, at least 24%, at least 28%, from 10 to 18%, from 12 to 16%, from 18 to 29%, from 20 to 27% or from 20 to 25% of the live weight of poultry fed with a synthetic oligosaccharide preparation, nutritional composition, animal feed premix or animal feed composition. In some embodiments, the average breast meat yield from an eviscerated poultry carcass fed a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition as described herein is at least 1%, at least 2% %, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 9%, at least 10% , at least 11%, at least 12%, 110%, 2-8% or 3-5% more than poultry fed a diet that does not include a synthetic oligosaccharide drug.

In some embodiments, the animal is poultry and the leg meat yield from the eviscerated poultry carcass is at least 5%, at least 7%, at least 8%, at least 9%, at least 10%, at at least 11%, at least 12%, from 5 to 14%, from 7 to 10%, from 7 to 15%, from 9 to 13% or from 9 to 11% of the live weight of birds receiving synthetic oligosaccharide a preparation, nutritional composition, animal feed premix or animal feed composition. In some embodiments, the yield of drumstick meat from an eviscerated poultry carcass fed with a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition as described herein is at least 1%, at least 2% %, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 9%, at least 10% , at least 11%, at least 12%, 1-10%, 2-8% or 3-5% more than poultry fed a diet that does not include a synthetic oligosaccharide preparation.

In some embodiments, the animal is poultry and the average leg meat yield from a gutted poultry carcass is at least 5%, at least 7%, at least 8%, at least 9%, at least 10% , at least 11%, at least 12%, 5 to 14%, 7 to 10%, 7 to 15%, 9 to 13% or 9 to 11% of the live weight of poultry treated with synthetic oligosaccharide preparation, nutritional composition, animal feed premix or animal feed composition. In some embodiments, the average drumstick meat yield from an eviscerated poultry carcass fed a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition as described herein is at least 1%, at least 1%.

- 54 044359 by at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 9%, by at least 10%, by at least

11%, at least 12%, 1-10%, 2-8% or 3-5% more than poultry fed a diet that does not include a synthetic oligosaccharide preparation.

In some embodiments, the animal is poultry and the boneless poultry breast fillet yield is at least 14%, at least 16%, at least 18%, at least 20%, at least 22%, at at least 24%, 14 to 16%, 18 to 30%, 20 to 28% or 20 to 26% of live weight for poultry fed with a synthetic oligosaccharide preparation, nutritional composition, animal feed premix or feed composition for animals. In some embodiments, the yield of breast fillet from a boneless poultry carcass treated with an oligosaccharide preparation, animal feed premix, or animal feed composition as described herein is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 9%, at least 10%, at least at least 11%, at least 12%, 1-10%, 2-8%, or 3-5% more than poultry fed a diet that does not include a synthetic oligosaccharide preparation.

In some embodiments, the animal is poultry and the average breast fillet yield from a boneless poultry carcass is at least 14%, at least 16%, at least 18%, at least 20%, at least 22%, at least 24%, 14 to 16%, 18 to 30%, 20 to 28% or 20 to 26% of live weight for poultry fed a synthetic oligosaccharide preparation, nutritional composition, animal feed premix or feed composition for animals. In some embodiments, the average yield of breast fillet from a boneless poultry carcass fed a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition as described herein is at least 1% by at least 1%. 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 9%, at least 10 %, at least 11%, at least 12%, 1-10%, 2-8% or 3-5% more than poultry fed a diet that does not include a synthetic oligosaccharide preparation.

In some embodiments, the animal is poultry and the boneless poultry leg meat yield is at least 6%, at least 8%, at least 10%, at least 12%, at least 14% , at least 16%, at least 18%, 6 to 18%, 8 to 16%, 12 to 21%, 14 to 19% or 14 to 17% of the live weight of poultry treated with synthetic oligosaccharide preparation, nutritional composition, animal feed premix or animal feed composition. In some embodiments, the yield of leg meat from a boneless poultry carcass fed a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition as described herein is at least 1% by at least 1%. 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 9%, at least 10 %, at least 11%, at least 12%, 1-10%, 2-8% or 3-5% more than poultry fed a diet that does not include a synthetic oligosaccharide preparation.

In some embodiments, the animal is poultry and the average leg meat yield from a boneless poultry carcass is at least 6%, at least 8%, at least 10%, at least 12%, at least 14 %, at least 16%, at least 18%, 6 to 18%, 8 to 16%, 12 to 21%, 14 to 19% or 14 to 17% of the live weight of poultry fed a synthetic oligosaccharide preparation, nutritional composition, animal feed premix or animal feed composition. In some embodiments, the average yield of leg meat from a boneless poultry carcass treated with an oligosaccharide preparation, animal feed premix, or animal feed composition as described herein is at least 1%, at least 2%, according to at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 9%, at least 10%, at least at least 11%, at least 12%, 1-10%, 2-8%, or 3-5% more than poultry fed a diet that does not include a synthetic oligosaccharide preparation.

In some embodiments, the animal is poultry and the poultry eviscerated carcass fat yield is at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4% , at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.2%, at least 1.4%, at least 1.6%, 0.1 to 2%, 0.2 to 1%, 0.5 to 2% or 0.3 to 0.7% of the live weight of poultry fed with a synthetic oligosaccharide preparation, nutritional composition, animal feed premix or animal feed composition. In some embodiments, the fat yield from an eviscerated poultry carcass treated with a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition as described herein is at least 1%, at least 1%. by 2%, by at least 3%, by at least 4%, by at least 5%, by at least 6%, by at least 8%, by at least 9%, by at least 10%, at least 11%, at least 12%, 1-10%, 2-8% or 3-5% more than poultry fed a diet that does not include a synthetic oligosaccharide preparation.

In some embodiments, the animal is poultry and the average fat yield from the eviscerated poultry carcass is at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4 %, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at at least 1.2%, at least 1.4%, at least 1.6%, from 0.1 to 2%, from 0.2 to 1%, from 0.5 to 2% or from 0, 3 to 0.7% of the live weight of poultry fed with a synthetic oligosaccharide preparation, nutritional composition, animal feed premix or animal feed composition. In some embodiments, the average fat yield from an eviscerated poultry carcass fed with a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition as described herein is at least 1%, at least 2% , by at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 9%, at least 10% , at least 11%, at least 12%, 1-10%, 2-8% or 3-5% more than poultry fed a diet that does not include a synthetic oligosaccharide preparation.

In some embodiments, the animal is poultry and the poultry eviscerated yield is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least at least 75%, at least 80%, at least 85%, at least 90%, from 50 to 95%, from 60 to 85% or from 65 to 75% of the live weight of poultry treated with a synthetic oligosaccharide preparation, a nutritional composition, an animal feed premix or an animal feed composition. In some embodiments, the yield of an eviscerated poultry carcass treated with a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition as described herein is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 9%, at least 10%, at least at least 11%, at least 12%, 1-10%, 2-8%, or 3-5% more than poultry fed a diet that does not include a synthetic oligosaccharide preparation.

In some embodiments, the animal is poultry and the average poultry evisceration yield is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least at least 75%, at least 80%, at least 85%, at least 90%, from 50 to 95%, from 60 to 85% or from 65 to 75% of the live weight of poultry treated with a synthetic oligosaccharide preparation, a nutritional composition, an animal feed premix or an animal feed composition. In some embodiments, the average yield of an eviscerated poultry carcass fed with a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition as described herein is at least 1%, at least 2%, by at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 9%, at least 10%, at least at least 11%, at least 12%, 1-10%, 2-8%, or 3-5% more than poultry fed a diet that does not include a synthetic oligosaccharide preparation.

Methods for deboning poultry carcasses are well known to those skilled in the field of poultry processing. It should be understood that meat obtained from poultry can be measured, for example, as the ratio of the mass of meat extracted to the final weight of the bird before processing. In some embodiments, the animal is a poultry, and the poultry is at least 35 days, at least 42 days, at least 6 weeks, at least 6.5 weeks old before the poultry is processed for evisceration production poultry carcasses, boneless poultry carcasses, white meat, breast fillets and breast meat, tray-packed chicken, whole poultry without giblets (WOG) or meat as described above.

In other embodiments, the animal is poultry and the animal product is eggs. In some embodiments, the animal is a pig and the pig product is pig meat (eg, which may be sold to consumers, processed into food, or consumed by humans). In some embodiments, the yield of the pork product is the yield obtained from an individual pig. In some embodiments, the average yield of a pork product is the yield obtained from each individual pig in a population of pigs, averaged across the population.

- 56 044359

In yet another embodiment, the average yield of a swine product is the total yield of a swine population divided by the number of individuals in the swine population.

In some embodiments, an animal or population of animals fed a synthetic oligosaccharide drug, nutritional composition, animal feed premix, or animal feed composition has a higher average daily gain, a higher average weekly gain, a higher total gain, a higher average total gain, or more a higher average yield of the animal product, or any combination thereof, than that of an animal or population of animals fed a diet that does not include a synthetic oligosaccharide preparation, but includes one or more antibiotics, one or more ionophores, soluble corn fiber, modified wheat starch or yeast mannan, or any combinations thereof.

One skilled in the art will appreciate that the maximum theoretical weight gain may be different for different types of animals, and may be different for different breeds of the same type of animal (eg, different types of broiler chickens or different types of pigs).

One skilled in the art will appreciate that the maximum theoretical weight gain may be different for different types of animals, and may be different for different breeds of the same type of animal (eg, different types of broiler chickens or different types of pigs).

In some embodiments, the animal is a poultry. In some embodiments, which may be combined with any of the above embodiments, poultry is a single poultry bird, while in other embodiments, poultry is a population of poultry birds. In other embodiments, the animal is a pig. In some embodiments, which may be combined with any of the above embodiments, the pig is an individual pig, while in other embodiments, the pig is a population of pigs.

G. Feed consumption

In some embodiments, providing an animal with a synthetic oligosaccharide drug, nutritional composition, animal feed premix, or animal feed composition as described herein results in increased average daily feed intake compared to an animal food that does not include the synthetic oligosaccharide drug.

Average daily feed intake (ADFI) refers to the average weight of feed consumed by an animal over a given period of time. In some embodiments, average daily feed intake is measured by distributing a known mass of feed to a group of a fixed number of animals, allowing the animals in the group to freely consume the distributed feed without restriction for a specified number of days, weighing the mass of unconsumed feed at the end of the time period, and calculating the average daily feed intake (ADFI) as the difference between the distributed feed mass minus the residual feed mass divided by the number of animals in the group and divided by the number of days in the period. In other embodiments, the average daily feed intake may be adjusted for any animals that die or are removed from the group using methods known to one of ordinary skill in the art.

In some embodiments, the animal is poultry and the animal feed composition is poultry feed, wherein the synthetic oligosaccharide drug, poultry feed premix, or poultry feed composition increases average daily feed intake by about 10%, or about 5 %, or from 1 to 10%, from 2 to 10%, from 3 to 10%, from 4 to 10%, from 5 to 10%, from 2 to 5%, from 2 to 6%, from 2 to 7% , from 2 to 8%, from 2 to 9% or from 1 to 5% when fed to poultry compared to poultry receiving a feed composition without a synthetic oligosaccharide preparation.

In some embodiments, the poultry is suffering from a disease or is being raised in an unfavorable environment where the synthetic oligosaccharide drug, poultry nutritional composition, poultry feed premix, or poultry feed composition increases average daily feed intake by about 30%, about 25%, about 20%, about 15%, about 10% or about 5%, or from 1 to 30%, from 5 to 30%, from 10 to 30%, from 5 to 20%, from 10 to 20% , from 1 to 20%, from 1 to 15%, from 1 to 10%, from 2 to 10%, from 3 to 10%, from 4 to 10%, from 5 to 10%, from 2 to 5%, from 2 to 6%, 2 to 7%, 2 to 8%, 2 to 9%, or 1 to 5% when fed to poultry compared to poultry fed the feed composition without the synthetic oligosaccharide preparation.

In some embodiments, which may be combined with the foregoing, the animal is a pig and the animal feed composition is a pig feed, wherein the oligosaccharide preparation, swine nutritional composition, swine feed premix, or swine feed composition increases average daily feed intake by about 15%, by about 10% or by about 5%, or from 1 to 15%, from 2 to 15%, from 3 to 15%, from 4 to 15%, from 5 and 15%, from 10 to 15 %, from 1 to 10%, from 2 to 10%, from 3 to 10%, from 4 to 10%, from 5 to 10%, from 2 to 5%, from 2 to 6%, from 2 to 7%, 2 to 8%, 2 to 9% or 1 to 5% when fed to pigs, compared to

- 57 044359 pigs receiving a feed composition without a synthetic oligosaccharide preparation.

In some embodiments, the pig has a disease or is raised in an unfavorable environment where the synthetic oligosaccharide drug, swine nutritional composition, swine feed premix, or swine feed composition increases average daily feed intake by about 40%, about 35%, about 30% , about 25%, about 20%, about 15%, about 10% or about 5%, or from 1 to 40%, from 5 to 40%, from 10 to 40%, from 15 to 40% , from 20 to 40%, from 25 to 40%, from 30 to 40%, from 1 to 30%, from 5 to 30%, from 10 to 30%, from 5 to 20%, from 10 to 20%, from 1 to 20%, from 1 to 15%, from 1 to 10%, from 2 to 10%, from 3 to 10%, from 4 to 10%, from 5 to 10%, from 2 to 5%, from 2 to 6%, 2 to 7%, 2 to 8%, 2 to 9%, or 1 to 5% when fed to pigs compared to pigs fed the feed composition without the synthetic oligosaccharide preparation.

Methods for enhancing the growth of an animal or animal population described herein include providing an oligosaccharide preparation, an animal feed premix, or an animal feed to an animal or animal population. The oligosaccharide preparation, animal feed premix, or animal feed may be provided in any suitable form to any suitable type of animal using any suitable feeding regimen to promote growth of the animal or population of animals.

VIII. Animals

The synthetic oligosaccharide preparation, nutritional composition, animal feed premix or animal feed composition may be provided to any suitable animal. In some embodiments, the animal is monogastric. It is generally believed that a monogastric animal has a single-chamber stomach. In other embodiments, the animal is a ruminant. It is generally accepted that ruminants have a multi-chambered stomach. In some embodiments, the animal is a ruminant in the preruminant phase. Examples of such ruminant animals in the preruminant phase are dairy calves.

In some embodiments, the animal is livestock. In some embodiments, the animal is a pet. In some embodiments, the animal is a poultry. Examples of poultry include chicken, duck, turkey, goose, quail or fighting cornish. In one embodiment, the animal is a chicken. In some embodiments, the poultry is a laying hen, broiler chicken, or turkey. In other embodiments, the animal is a mammal, including, for example, cow, pig, goat, sheep, deer, bison, rabbit, alpaca, llama, mule, horse, reindeer, buffalo, yak, guinea pig, rat, mouse, alpaca , dog, cat or person. In one embodiment, the animal is a cow. In another embodiment, the animal is a pig.

The animal feed composition can also be used in aquaculture. In some embodiments, the animal is an aquatic animal. Examples of aquatic animals may include trout, salmon, grouper, tilapia, shrimp, oyster, mussel, clam, lobster or crayfish. In one embodiment, the animal is a fish.

In some embodiments, the animal is a fish (e.g., salmon, tilapia, tropical fish), poultry (e.g., chicken, turkey), marine organism (e.g., shrimp), sheep, cow, cattle, buffalo, bison, pig (e.g. growing piglets, piglets in the first/second phase of fattening, cat, dog, rabbit, goat, guinea pig, donkey, camel, horse, pigeon, ferret, gerbil, hamster, mouse, rat, bird or human.

IX. Application

In some embodiments, use includes providing a synthetic oligosaccharide preparation, nutritional composition, or animal feed composition described herein to an animal so that the animal can consume the synthetic oligosaccharide preparation, nutritional composition, or animal feed composition as desired. In such embodiments, the animal consumes a portion of the synthetic oligosaccharide drug, nutritional composition, or animal feed composition.

The synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition may be provided to the animal on any suitable schedule. In some embodiments, the animal is provided with a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition daily, weekly, monthly, every other day, for at least three days a week, or at least seven days every month. In some embodiments, the oligosaccharide preparation, animal feed premix, or animal feed composition is administered to the animal during certain phases of the diet.

For example, some animals are given a starter diet between 0 and 14 days of age. In other embodiments, the animal is given a growth diet at 15 to 28 days of age, 15 to 35 days of age, or

- 58 044359 from 15 to 39 days. In other embodiments, the animal is given a finishing diet at 29 to 35 days, 36 to 42 days, or 40 to 46 days of age.

In some embodiments, the synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition is provided to the animal during the starter phase of the diet, the growth phase of the diet, or the finishing phase of the diet, or any combinations thereof.

In some embodiments, the animal is a poultry and the poultry is fed a starter diet from 0 to 15 days of age, a grower diet from 16 to 28 days of age, and a finisher diet from 29 to 35 days of age. In other embodiments, the animal is a poultry and the poultry is fed a starter diet from 0 to 14 days of age, a grower diet from 15 to 35 days of age, and a finisher diet from 36 to 42 days of age. In other embodiments, the animal is a poultry and the poultry is fed a starter diet from 0 to 14 days of age, a grower diet from 15 to 39 days of age, and a finisher diet from 20 to 46 days of age.

In some embodiments, the synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition is provided to poultry during the starter phase of the diet, the growth phase of the diet, or the finishing phase of the diet, or any combination thereof.

The oligosaccharide preparations described herein can be fed to individual animals or populations of animals. For example, in one embodiment, where the animal is poultry, the oligosaccharide preparations can be fed to an individual poultry or group of poultry.

The synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition may be provided to the animal in any suitable form, including, for example, solid form, liquid form, or a combination thereof. In some embodiments, the oligosaccharide preparation or animal feed composition is a liquid, such as a syrup or solution. In other embodiments, the oligosaccharide preparation, animal feed premix, or animal feed composition is a solid, such as a granule or powder. In still other embodiments, the oligosaccharide preparation, animal feed premix, or animal feed composition may be provided to the animal in both liquid and solid components, such as a mash.

Examples

Example 1. Synthesis of glucogalactooligosaccharide drug

Synthesis of the glucogalacto-oligosaccharide drug was carried out in a three-liter reaction vessel using catalyst loading, reaction time, and reaction temperature that were chosen to ensure suitable kilogram-scale production.

D-glucose monohydrate (825.16 g), D-lactose monohydrate (263.48 g) and 2-pyridine sulfonic acid (1.0079 g, Sigma-Aldrich, St. Louis, USA) were added to a three-liter three-neck round bottom flask and a central neck made of ground glass 29/42 and two side necks made of ground glass 24/40. A 133 mm Teflon stirring paddle was attached to the glass stirring rod using PTFE tape. The stirrer rod was secured through the center point using a Teflon carrier adapter and attached to an overhead high torque mechanical mixer via a flexible connector. The flask was secured inside a hemispherical electric heating mantle, controlled by a temperature control unit through a J-type thermocouple rod inserted through a rubber septum in one of the side ports. The thermocouple tip was adjusted so that it was in the reaction mixture with a gap of a few mm above the mixing element. The secondary temperature probe connected to the auxiliary temperature sensor was also inserted and secured in the same manner. The second side port of the flask was equipped with a reflux condenser cooled by a water-glycol mixture maintained below 4°C by a recirculating cooling bath.

The reaction mixture was gradually heated to 130°C with continuous stirring at a stirring speed of 80-100 rpm. When the reaction mixture reached 120°C, the reflux condenser was placed in distillation configuration, with the distilled product collected in a 250 mL round bottom flask placed in an ice bath. The mixture was kept at 130°C with continuous stirring for 6 h, after which the thermocouple unit was turned off. The distillation apparatus was removed, and 390 g of distilled water at 60°C was gradually added to the three-neck flask. The resulting mixture was left to stir at 40 rpm for 10 hours. Approximately 1250 g of a viscous light amber colored material was collected and its concentration was 71.6°Brix based on the refractive index.

The final water content of the reactor product was measured by Karl Fischer titration of a representative aliquot of the reactor contents taken at the end of the reaction. At a reaction temperature of 130°C, the water content in the reaction product was determined to be 5.8 wt.% water, not calculated on a dry basis.

- 59 044359

Example 2. Synthesis of a glucooligosaccharide drug

Synthesis of the gluco-oligosaccharide drug was carried out in a three-liter reaction vessel using catalyst loading, reaction time, and reaction temperature that were selected to allow suitable kilogram-scale production.

D-glucose monohydrate (1150 g) was added to a three-liter, three-neck round bottom flask with one 29/42 ground glass center neck and two 24/40 ground glass side necks. A 133 mm Teflon stirring paddle was attached to the glass stirring rod using PTFE tape. The stirrer rod was secured through the center point using a Teflon carrier adapter and attached to an overhead high torque mechanical mixer via a flexible connector. The flask was secured inside a hemispherical electric heating mantle, controlled by a temperature control device, through a J-type thermocouple rod inserted through a rubber septum in one of the side ports. The thermocouple tip was adjusted so that it was in the reaction mixture with a gap of a few mm above the mixing element. A secondary temperature probe connected to the secondary temperature sensor was also inserted and secured in the same manner. The second side port of the flask was equipped with a reflux condenser cooled by a water-glycol mixture maintained below 4°C by a recirculating cooling bath.

The reaction mixture was gradually heated to 130°C with continuous stirring at a stirring speed of 80-100 rpm. When the reaction temperature increased to 120-130°C, (+)-camphor-10-sulfonic acid (1.16 g, Sigma-Aldrich, St. Louis) was added to the three-neck flask and the apparatus was switched from reflux to the distillation configuration with round bottom collection flask placed in an ice bath. This setup was maintained for 1.5 h, after which the thermocouple unit was turned off, the distillation apparatus was removed, and 390 g of distilled water at 23°C was gradually added to the three-neck flask. The resulting mixture was left stirring at 40 rpm for 10 h until collection. Approximately 1300 g of dark amber viscous material was collected and the concentration was 72.6° Brix.

Example 3. Synthesis of glucogalactomannooligosaccharide drug

Synthesis of the glucogalactomannooligosaccharide drug was carried out in a three-liter reaction vessel using catalyst loading, reaction time, and reaction temperature that were chosen to ensure suitable kilogram-scale production.

The glucogalactomannooligosaccharide preparation was prepared as two separate components, synthesized in separate reaction vessels, which were collected independently. Each synthesis used different starting reagents but followed the same procedures and methods until completion. The final glucogalactomannooligosaccharide preparation was a homogeneous syrup obtained by mixing both synthesis products.

To synthesize the first component, 990.54 g of glucose monohydrate, 105.58 g of lactose monohydrate and 1.00 g of 2-pyridine sulfonic acid were added to a three-liter three-neck round-bottom flask with a central ground neck 29/42 and side ground necks 24/40. A 133mm Teflon stirring paddle was attached to a 440mm diameter glass stirrer using PTFE tape. The stirrer rod was secured through the center point using a Teflon carrier adapter and attached to an overhead high torque mechanical mixer via a flexible connector. The flask was placed in a hemispherical electric heating mantle, controlled by a temperature control device through a J-type thermocouple rod inserted through a rubber septum into one of the side ports. The thermocouple tip was adjusted so that it was in the reaction mixture with a gap of a few mm above the mixing element. The secondary temperature probe connected to the auxiliary temperature sensor was also inserted and secured in the same manner. The second side port of the flask was equipped with a reflux condenser cooled by a water-glycol mixture maintained below 4°C by a recirculating cooling bath.

The reaction mixture was gradually heated to 130°C with continuous stirring at a stirring speed of 80-100 rpm. Once the temperature control chamber reached 120°C to 130°C, the unit was switched from reflux to a distillation configuration with a round-bottom collection flask placed in an ice bath. This setting was maintained for approximately 6 hours and 10 minutes, after which the heating mantle was turned off, the distillation apparatus was removed and 390 g of distilled water at 60° C. was gradually added to the three-neck flask. The resulting mixture was left stirring at 40 rpm for 10 h until collection. Approximately 1250 g of a viscous, light amber colored material was collected and measured at 73.1° Brix by refractive index.

To synthesize the second component, 825.04 g of glucose monohydrate, 251.16 g of pure mannose from wood, 25.10 g of distilled water and 1.00 g of 2-pyridine sulfonic acid were added to a three-liter three-neck round bottom flask with one central 29/42 ground neck. on the sides of which there are two polished necks 24/40. The rest of the synthesis of the second component

- 60 044359 was carried out using the same procedure and methods as for the first component until collection. Approximately 1250 g of dark amber viscous material was collected and the concentration was 72.3° Brix.

The first and second components were completely transferred into a suitable size HDPE container and thoroughly mixed by hand until homogeneous. The final syrup mixture weighed approximately 2.5 kg, was dark amber in color, was viscous and had a concentration of approximately 72° Brix.

Example 4. Synthesis of glucomannooligosaccharide drug

Synthesis of the gluco-oligosaccharide drug was carried out in a three-liter reaction vessel using catalyst loading, reaction time, and reaction temperature that were selected to allow suitable kilogram-scale production.

The glucomannooligosaccharide preparation was prepared as two separate components, synthesized in separate reaction vessels, which were collected independently. Each synthesis used different starting reagents but followed the same procedures and methods until completion. The final glucomannooligosaccharide preparation was a homogeneous syrup obtained by mixing both synthesis products.

To synthesize the first component, 1264.80 g of glucose monohydrate was added to a three-liter, three-neck round bottom flask with one central 29/42 ground neck surrounded by two 24/40 ground necks. A 133 mm Teflon stirring paddle was attached to a 440 mm diameter glass stirrer using PTFE tape. The stirrer rod was secured through the center point using a Teflon carrier adapter and attached to an overhead high torque mechanical mixer via a flexible connector. The flask was placed in a hemispherical electric heating mantle, controlled by a temperature control device through a J-type thermocouple rod inserted through a rubber septum in one of the side ports. The thermocouple tip was adjusted so that it was in the reaction mixture with a gap of a few mm above the mixing element. The secondary temperature probe connected to the auxiliary temperature sensor was also inserted and secured in the same manner. The second side port of the flask was equipped with a reflux condenser cooled by a water-glycol mixture maintained below 4°C by a recirculating cooling bath.

The reaction mixture was gradually heated to 130°C with continuous stirring at a stirring speed of 80-100 rpm. Once the temperature control chamber reached 120 to 130°C, 1.15 g of (+)-camphor-10-sulfonic acid was added to the three-neck flask and the unit was switched from reflux to the distillation configuration with a round bottom flask to collect placed in an ice bath. This setting was maintained for approximately 1 h, after which the thermocouple unit was turned off, the distillation apparatus was removed, and 390 g of distilled water at 23 °C was gradually added to the three-neck flask. The resulting mixture was left stirring at 40 rpm for 10 h until collection. Approximately 1350 g of viscous, light amber colored material was collected and the concentration was 71.8° Brix.

To synthesize the second component, 949.00 g of glucose monohydrate, 288.00 g of pure mannose from wood, 27.94 g of distilled water and 1.15 g of 2-pyridine sulfonic acid were added to a three-liter three-neck flask with one central polished neck 29/42, according to on the sides of which there are two polished necks 24/40. The remainder of the synthesis of the second component followed the same procedure and methods as for the first component until collection, except that (+)-camphor-10-sulfonic acid was not added since the reflux condenser was switched to the distillation configuration, and The resulting setup was maintained for approximately 6 hours. Approximately 1350 g of a viscous dark amber colored material was collected and its concentration was 72.0° Brix.

The first and second components were completely transferred into a suitable size HDPE container and thoroughly mixed by hand until homogeneous. The final syrup mixture weighed approximately 2.7 kg, was dark amber in color, was viscous and, as determined by refractive index, had a concentration of approximately 72° Brix.

Example 5. Synthesis of glucomannooligosaccharide drug

Kilogram-scale production of the oligosaccharide drug was accomplished in a three-liter reaction vessel using catalyst loading, reaction time, and reaction temperature found to be suitable for 1-kg scale production.

The glucomannooligosaccharide preparation was prepared as two separate components, synthesized in separate reaction vessels, which were collected independently. Each synthesis used different starting reagents but followed the same procedures and methods until completion. The final glucomannooligosaccharide preparation was a homogeneous syrup obtained by mixing both synthesis products.

To synthesize the first component, 1261.00 g of glucose monohydrate and 1.15 g of 2-pyridine sulfonic acid were added to a three-liter, three-neck round bottom flask with one central 29/42 ground neck surrounded by two 24/40 ground necks. A 133 mm Teflon stirring paddle was attached to a 440 mm diameter glass stirrer using PTFE tape. The stirrer rod was secured through the center point using a Teflon carrier adapter and attached to an overhead high torque mechanical mixer via a flexible connector. The flask was secured inside a hemispherical electric heating mantle, controlled by a temperature control device through a J-type thermocouple rod inserted through a rubber septum in one of the side ports. The thermocouple tip was adjusted so that it was in the reaction mixture with a gap of a few mm above the mixing element. The secondary temperature probe connected to the auxiliary temperature sensor was also inserted and secured in the same manner. The second side port of the flask was equipped with a reflux condenser cooled by a water-glycol mixture maintained below 4°C by a recirculating cooling bath.

The reaction mixture was gradually heated to 130°C with continuous stirring at a stirring speed of 80-100 rpm. Once the temperature control unit reading reached 120 to 130°C, the apparatus was switched from a reflux condenser to a distillation configuration with a round-bottom collection flask placed in an ice bath. This setting was maintained for approximately 6 hours, after which the thermocouple unit was turned off, the distillation apparatus was removed and 390 g of distilled water at 23°C was gradually added to the three-neck flask. The resulting mixture was left stirring at 40 rpm for 10 h until collection. Approximately 1250 g of viscous light yellow material was collected and the concentration was 73.5° Brix.

To synthesize the second component, 949.00 g of glucose monohydrate, 288.00 g of pure mannose from wood, 28.94 g of distilled water and 1.15 g of 2-pyridine sulfonic acid were added to a three-liter three-neck flask with one central polished neck 29/42, according to on the sides of which there are two polished necks 24/40. The remainder of the synthesis of the second component followed the same procedure and methods as for the first component until collection. Approximately 1250 g of viscous, dark amber colored material was collected and measured at 73.3° Brix.

The first and second components were completely transferred into a suitable size HDPE container and thoroughly mixed by hand until homogeneous. The final syrup mixture weighed approximately 2.5 kg, was dark amber in color, viscous and had a concentration of approximately 73° Brix.

Example 6. Synthesis of glucogalactooligosaccharide drug

Kilogram-scale production of the oligosaccharide drug was carried out in a three-liter reaction vessel using catalyst loadings, reaction times, and reaction temperatures that were found to be suitable for 1-kg scale production.

A 3-L three-neck flask was equipped with an overhead mixer connected through a 10 mm diameter glass stirrer to a 14 cm crescent-shaped mixing element. The mixing element was located at a distance of approximately 5 mm from the walls of the flask. The flask was heated by a hemispherical electric heating mantle controlled by a temperature control unit connected to a rod-shaped thermocouple probe inserted into the reaction flask. The thermocouple probe was placed so that there was a gap of 5-10 mm above the mixing element. The flask was charged with 576 g of food grade dextrose monohydrate and 577 g of food grade D-galactose monohydrate and heated to approximately 115° C. to obtain a molten sugar syrup. After obtaining the syrup, the flask was equipped with a jacketed reflux condenser, cooled to 4°C by circulating cooled glycol/water and temperature. 31 g of Dowex Marathon C resin (moisture content 0.48 g H ₂ O/g resin) was added to the mixture to form a stirred slurry. The condenser was placed in distillation configuration and the slurry was heated to 145°C.

A stirring speed of approximately 80 rpm and a temperature of 145°C were maintained for 3.8 hours, after which the temperature on the temperature control unit was reduced to 80°C and 119 ml of deionized water at a temperature of 60°C was gradually added to the flask to obtain dark amber syrup containing residual Dowex resin. The resulting suspension was further diluted to 60° Brix, cooled to room temperature and vacuum filtered through a 0.45 µm filter to remove resin. 1200 g of light amber syrup with a concentration of 60° Brix were obtained.

Example 7. Synthesis of a glucooligosaccharide drug

Kilogram-scale production of the oligosaccharide drug was accomplished in a three-liter reaction vessel using catalyst loadings, reaction times, and reaction temperatures that were found to be suitable for 1-kg scale production.

A three-neck flask with a capacity of 3 L was equipped with an overhead mixer connected through a glass stirrer with a diameter of 10 mm to a crescent-shaped mixing element measuring 14 cm. The mixing element was placed at a distance of approximately 5 mm from the walls of the flask. The flask was heated by a hemispherical electric heating mantle controlled by a temperature control unit connected to a rod-shaped thermocouple probe inserted into the reaction flask. The thermocouple probe was placed so that there was a gap of 5-10 mm above the mixing element. The flask was gradually charged with 1148 g of food grade dextrose monohydrate and heated to approximately 115°C to obtain

- 62 044359 melted sugar syrup. After obtaining the syrup, the flask was equipped with a jacketed distillation condenser, cooled to 4°C by circulating cooled glycol/water. The reaction temperature was gradually increased to 145°C. Once the temperature had been reached and stabilized, 31 g of Dowex Marathon C resin (moisture content 0.48 g _H2O /g resin) was added to the mixture and the stirring speed was maintained at approximately 80 rpm and the temperature at 145°C for 3.8 hours.

After 3.8 hours, the temperature control was lowered to 80°C and 119 mL of 60°C deionized water was gradually added to the flask to produce a dark amber syrup containing residual Dowex resin. The resulting suspension was further diluted to 60° Brix, cooled to room temperature and vacuum filtered through a 0.45 µm filter to remove resin. 1113 g of dark amber-colored gluco-oligosaccharide syrup with a concentration of 60° Brix was obtained.

Example 8 One-pot synthesis of oligosaccharide drugs

A one-pot (one-component) synthesis of the oligosaccharide from Example 3 was demonstrated on a 300 g scale in a one-liter reaction vessel using catalyst loadings, reaction times and reaction temperatures that were found to be suitable for a one-pot reaction.

g food grade D-glucose monohydrate from corn, 37.50 g food grade D-mannose from wood, 15.60 g food grade D-lactose monohydrate, 3.96 g distilled water and 0.270 g 2-pyridine sulfonic acid (Sigma-Aldrich , St. Louis) was added to a one-liter, three-neck round bottom flask with one central 29/42 ground neck surrounded by two 24/40 ground necks. A Teflon stirring paddle was attached to a 220mm glass stirring shaft using PTFE tape. The stirrer rod was secured through the center point using a Teflon carrier adapter and attached to an overhead high torque mechanical mixer via a flexible connector. The flask was secured inside a hemispherical electric heating mantle, controlled by a temperature control device through a J-type thermocouple rod inserted through a rubber septum in one of the side ports. The thermocouple tip was adjusted so that it was in the reaction mixture with a gap of a few mm above the mixing element. The secondary temperature probe connected to the auxiliary temperature sensor was also inserted and secured in the same manner. The second side port of the flask was equipped with a reflux condenser cooled by a water-glycol mixture maintained below 4°C by a recirculating cooling bath.

The reaction mixture was gradually heated to 130°C with continuous stirring at a stirring speed of 80-100 rpm. Once the temperature control chamber reached 120 to 130°C, the unit was switched from reflux to a distillation configuration with a round-bottom collection flask placed in an ice bath. The mixture was maintained at 130°C with continuous stirring for approximately 5 hours 40 minutes, after which the heating mantle and distillation apparatus were removed. Approximately 40 g of distilled water at 23°C was gradually added to a three-neck flask. The resulting mixture was left stirring at 40 rpm for 10 h until collection. Approximately 389 g of dark amber viscous material was collected and the concentration was 67.0° Brix. Compliance with the oligosaccharide drug from Example 3 was confirmed by SEC chromatography and 2D HSQC NMR spectroscopy.

Example 9. Characteristics of oligosaccharide preparations

The methods and procedures from Examples 1-8 were used to prepare repeat batches and mixtures of oligosaccharides from Examples 1-7. When preparing various batches from examples 9.1-9.7, both multi-reactor (multicomponent) and single-reactor versions of the corresponding synthesis schemes were used. The resulting materials were analyzed by HPLC size exclusion chromatography (SEC) to characterize molecular weight distribution, LC-MS/MS analysis to quantify DP2 anhydrosaccharide content, and 2D 1H,13C-HSQC NMR to determine the molecular structure of the corresponding oligosaccharide drugs. The materials were prepared as follows.

Example 9.1. Eleven batches of the oligosaccharide preparation from Example 1 were prepared and mixed in four separate batches to obtain oligosaccharide preparation 9.1.

Example 9.2. Seven batches of the oligosaccharide preparation from Example 2 were prepared and mixed in two separate batches to obtain oligosaccharide preparation 9.2.

Example 9.3. Twelve batches of the oligosaccharide preparation from Example 3 were prepared and mixed in five separate batches to obtain oligosaccharide preparation 9.3.

Example 9.4. Four batches of the oligosaccharide preparation from Example 4 were prepared and mixed into one batch to obtain oligosaccharide preparation 9.4.

Example 9.5. Four batches of the oligosaccharide preparation from Example 5 were prepared and mixed into one batch to obtain oligosaccharide preparation 9.5.

Example 9.6. Two batches of the oligosaccharide preparation from Example 6 were prepared and mixed into one batch to obtain oligosaccharide preparation 9.6.

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Example 9.7. Two batches of the oligosaccharide preparation from Example 7 were prepared and mixed into one batch to obtain oligosaccharide preparation 9.7.

Additional structural variants of the oligosaccharide drugs from Examples 1-7 were synthesized on a 300 gram scale using the methods from Examples 1-7, but with variations in the starting sugar compositions, acid, acid load, reaction time and temperature. Oligosaccharide preparations were synthesized as follows:

Example 9.8. 300 g of sucrose, 3 g of phosphoric acid and 27 g of water were reacted at 125°C for approximately 1 hour to obtain a dark brown oligosaccharide syrup, which was then diluted to 60° Brix with distilled water.

Example 9.9. 270 g of glucose, 30 g of sucrose, 0.3 g of phenylphosphonic acid and 27 g of water were reacted at 130°C for 1 to 4 hours to obtain a dark brown oligosaccharide syrup, which was then diluted to 60° Brix with distilled water.

Example 9.10. 225 g glucose, 75 g lactose, 3 g butylphosphonic acid and 27 g water were reacted at 130°C for 1 to 4 hours to produce a dark amber oligosaccharide syrup, which was then diluted to 60° Brix with distilled water.

Example 9.11. 225 g of glucose, 75 g of lactose, 3 g of phenylphosphonic acid and 27 g of water were reacted at 130°C for 1 to 5 hours to obtain a dark amber oligosaccharide syrup, which was then diluted to 60° Brix with distilled water.

Example 9.12. 270 g of glucose, 30 g of lactose, 3 g of phenylphosphinic acid and 27 g of water were reacted at 130°C for 3 to 5 hours to obtain a dark brown oligosaccharide syrup, which was then diluted to 60° Brix with distilled water.

Example 9.13. 300 g glucose, 3 g phenylphosphinic acid and 27 g water were reacted at 130°C for 1 to 3 hours to produce a dark amber oligosaccharide syrup, which was then diluted to 60° Brix with distilled water.

Example 9.14. 300 g of glucose, 2 g of propionic acid and 27 g of water were reacted at 130°C for 1 to 4 hours to obtain an amber oligosaccharide syrup, which was then diluted to 60° Brix with distilled water.

Example 9.15. 300 g of glucose, 0.15 g of 8-hydroxy-5-quinoline sulfonic acid hydrate and 27 g of water were reacted at 130°C for 2-4 hours to obtain an succinic oligosaccharide syrup, which was then diluted to 60° Brix with distilled water.

In the above reactions, all masses refer to the masses of the pure components, and the total mass of reacting water included any additional water provided by moisture content and/or water from hydration of the reacting sugars.

Characteristics of oligosaccharide preparations

The resulting materials were analyzed by HPLC size exclusion chromatography (SEC) to characterize molecular weight distribution, LC-MS/MS analysis to quantify DP2 anhydrosaccharide content, and 2D 1H, ^13C -HSQC NMR to determine the molecular structure of the corresponding oligosaccharide drugs.

Analysis of molecular weight of polymers using HPLC

The number average molecular weight (MWn) and weight average molecular weight (MWw) of the oligosaccharide preparations from Examples 9.1-9.7 were determined using HPLC. SEC analysis was performed on an Agilent 1100 series HPLC with refractive index determination using an Agilent PL aquagel-OH 20 column at 40°C with distilled water as the mobile phase at 0.45 mL/min. Retention time calibration to MW was performed using standard solutions of known molecular weight, and standard prior art methods were used to determine various distribution properties from the SEC chromatogram. The MWn and MWw values of oligosaccharide preparations from many series are shown in the table below. 1.

Table 1. MWn and MWw of oligosaccharide drugs

Oligosaccharide drug MWn (g/mol) MWw (g/mol) 9.1 719 ± I 1.063 ±23 9.2 808 ±30 1.336 ± 122 9.3 757 ± 15 1.186 ±49 9.4 761 1,196 9.5 755 1.177 9.6 505 709 9.7 762 ± 12 1.154 ± 14

Anhydro-DP2 Analysis by LC-MS/MS

The DP2 anhydro-oligosaccharide content of the oligosaccharide preparations was determined by LC-MS/MS using a Capcell Pak NH2 column (Shiseido; 250 x 4.6 mm, 5 μm) at a flow rate of 1 mL/min under isocratic water/acetonitrile 35/65 conditions. Before MS, the flow was split 1:4 and a feed flow of 50 μL of 0.05% NH4OH was added to enhance ionization. For MS detection, an ESI probe in negative mode was used, and the multiple reaction monitoring (MRM) method allowed for targeted analysis.

The anhydro-DP2 content of the oligosaccharide preparations was first determined relative to the content of the oligosaccharide preparation from Example 9.7 as a reference composition. The absolute anhydro-DP2 content of the reference oligosaccharide preparation from Example 9.7 was then determined by HPLC-MS/MS to be approximately 10%, and the anhydro-DP2 content of the oligosaccharide preparations from Examples 9.1-9.6 was then calculated. The relative and absolute content of DP2 was determined as described in Table. 2.

Table 2. Content of anhydro-PD2 in oligosaccharide preparations with several series

Oligosaccharide drug Relative content of anhydro-PR2, in% compared to example 9.7 Anhydro-PR2 content (gram anhydro-PR2/gram total DP2) 9.1 53% 5.3% 9.2 14% 1.4% 9.3 57% 5.7% 9.4 53% 5.3% 9.5 33% 3.3% 9.6 50% 5.0% 9.7 100% 10.0%

Molecular fingerprinting by 2D 1H, ¹³ C-HSQC NMR

The molecular structures of the oligosaccharide drugs from Example 9 were characterized using 2D 1H, ¹³ C-HSQC NMR spectroscopy. Samples were prepared by drying 125 mg (dry matter basis) of the oligosaccharide preparation at 40°C and redissolving in D2O containing 0.1% acetone. NMR spectra were obtained at 300 K on either a Bruker Avance 400 MHz NMR spectrometer or a Bruker Avance III 600 MHz NMR spectrometer equipped with a cryogenically cooled 5 mm TCI probe. In fig. 1 shows an illustrative 2D 1H, ^13C HSQC NMR spectrum of oligosaccharide drug 9.7.

Anomeric region of the spectrum 1H, ¹³ C-HSQC, F2 (1H) = 4.2-6.0 ppm. and F1 ( ¹³ C) = 90-120 ppm. was used to fingerprint the bond distribution of oligosaccharide drugs. Each peak in the anomeric region was integrated and its relative abundance was determined relative to that of the entire anomeric region. 2D 1H, ¹³ C HSQC fingerprinting was performed on four series of oligosaccharide preparation 9.1.

Table 3. Relative abundance of peaks F1 and F2 from oligosaccharide preparation 9.1

F2 (ppm) F1 (ppm) AUC (Mean ± SEM) 5.43 92.42 0.4% ± 0.3% 5.44 102.07 0.4% ± 0.1% 5.43 90.05 0.5% ± 0.2% 5.40 100.22 1.6% ± 0.4% 5.37 98.33 0.7% ± 0.4% 5.35 99.70 2.7% ± 0.6% 5.33 96.53 0.3% ± 0.2% 5.24 100.86 0.5% ± 0.2% 5.22 92.71 20.2% ± 3.9% 5.21 102.45 0.5% ± 0.4% 5.18 93.86 0.9% ± 0.4% 5D7 96.01 0.4% ± 0.1% 5.09 96.88 0.6% ± 0.3% 5.03 108.49 0.4% ± 0.2% 5.02 109.16 0.4% ± 0.4% 4.98 99.19 0.6% ± 0.3% 4.95 98.51 30.6% ±4.1% 4.86 98.53 0.7% ± 0.5% 4.79 96.84 0.6% ± 0.3% 4.71 103.48 2.5% ± 0.7% 4.64 103.56 0.8% ± 0.4% 4.63 102.49 0.7% ± 0.5% 4.62 104.56 1.4% ± 0.4% 4.57 97.07 1.6% ± 0.3% 4.50 103.30 25.9% ± 2.2% 4.45 103.56 2.4% ± 1.3%

Example 10. Determination of anhydrous sugar subunits in an oligosaccharide preparation

The relative abundance of anhydrous sugar subunits in the oligosaccharide preparations of Example 9 was determined using MALDI-MS on a Bruker Ultraflex instrument. Samples were dissolved in water until

- 65 044359 concentration of 10 mg/ml, of which 5 μl was mixed with a matrix solution (30 mg/ml DHB in 80% ethanol and water in a ratio of 1:10). The plates were prepared by applying 1 μL of analyte solution to the target plate and allowed to dry in ambient air. In some cases, samples were recrystallized using 1 μL ethanol before MS analysis.

Fig. 2 presents an exemplary MALDI spectrum of the oligosaccharide preparation from Example 9. The sugar anhydrosubunits are clearly observed as shifted peaks, shifted by -18 g/mol relative to its corresponding major parent DP. Shifted peaks are observed at all DP values, indicating that anhydrous sugar subunits are found at all oligosaccharide sizes. The relative peak intensity of the anhydro subunits as determined was approximately 10% of the total peak intensity for each DP value, from which the relative abundance of all anhydro subunits was determined to be approximately 10%. Fig. 25A and 25B illustrate MALDI spectra of the oligosaccharide preparation from Example 2. Anhydrous sugar subunits are observed at each DP level with relative intensities in the range of 5-10%.

Example 11. Characteristics of anhydrosubunits of an oligosaccharide drug

The anhydrosaccharide subunits of the oligosaccharide preparations from Example 9 were characterized using a combination of LC-MS, GC-MS, LC-MS/MS and NMR methods.

Characteristics of anhydro-DPI components

The anhydrous component DP1 from the oligosaccharide preparation of Example 9 was isolated using preparative liquid chromatography. The isolated anhydro-DP1 component was obtained for NMR by dissolving it in 0.75 ml of D ₂ O. In FIG. 3 shows an exemplary 1H-NMR ID spectrum of an anhydro DP1 fraction isolated from the oligosaccharide of Example 9, and FIG. 4 shows an illustrative APT ¹³ C-NMR spectrum of the same isolated fraction of anhydro DP1.

Using the following peak assignments in the table. 4, the ratio of 1,6-anhydro-beta-D-glucofuranose to 1,6-anhydro-beta-D-glucopyranose as determined by NMR was 2:1.

Table 4. NMR Peak Assignments

1,6-Anhydro-beta-Oglucofuranose 1,6-Anhydro-beta-Oglucopyranose # 'N (m.d.) ¹³ C (ppm) 'N (m.d.) ¹³ C (ppm) 1 5.33 101.9 5.01 104.4 2 3.40 70.6 4.37 79.8 3 3.56(ov) ^a 73.0 4.27 78.3 4 3.56(ov) ^a 71.3 4.38 80.6 5 4.50 76.7 3.74 64.1 6 3.97;3.64 65.7 4.14; 3.72 66.7

^a Ov means overlapping signal.

Characteristics of anhydro-PD2 components

The anhydro-DP2 content of the oligosaccharide preparations from Example 9 was determined using a combination of LC-MS, GC-MS, LC-MS/MS and NMR methods. The content of anhydro-DP2 in the oligosaccharide preparations from Example 9 was determined using GC-MS and LC-MS/MS analyses. Gas chromatography was performed using a 30 m x 0.25 mm fused silica column containing HP-5MS stationary phase with helium at a constant pressure of 21.57 psi as carrier gas. Aliquots were pre-derivatized by acetylation by dissolving 20 mg of sample in 0.5 mL of pyridine with 0.5 mL of acetic anhydride for 30 min at 60°C. 1 μL samples were injected at 300°C with an oven temperature program starting at 70°C and gradually increasing by 10°C per minute to 315°C. Detection was performed on an Agilent 5975C MSD with an electron energy of 70 eV.

Fig. Figure 5 shows an enlargement of the GC-MS chromatogram for oligosaccharide drug 9.7. TIC and XIC plots (m/z 229) demonstrate that the DP2 and anhydro-DP2 components are clearly separated. Fig. 30A30B, 31A-31B, 32A-32B, and 33A-33B illustrate the presence of the DP1, anhydro-DP1, DP2, and anhydro-DP2 fractions as determined by GC-MS in the oligosaccharide preparation of Example 1, Example 3, Example 4, and Example 7, respectively. As shown in FIG. 30A-30B, 31A-31B, 32A-32B and 33A-33B, the anhydro-DP1 and DP1 fractions have retention times of approximately 12-17 minutes, and the anhydro-DP2 and DP2 fractions have retention times of approximately 2-25 minutes.

Fig. 36 illustrates MALDI-MS spectra comparing the oligosaccharide formulation of Example 9 at different laser energies. The relative abundance of the signals remained virtually unchanged, indicating that laser ionization does not result in water loss. Consequently, the presence of anhydrosaccharide subunits in the oligosaccharide preparation has been proven.

Example 12. Observation of subunits from caramelization in an oligosaccharide preparation

An oligosaccharide drug comprising the 5-hydroxymethylfurfural subunit from caramelization was demonstrated by a combination of HPLC and 2D 1H, ^13C HSQC NMR analyses. A 50 mg aliquot of the oligosaccharide drug from Example 9 was dissolved in 0.8 ml D ₂ O. The resulting 2D spectrum

- 66 044359 ¹ H, ¹³ C HSQC was analyzed for the presence of a glycosidic bond between 5-hmf and the anomeric carbon of glucose with the following peak assignments: ¹ H-NMR: δ= 9.39 ppm. (CHO, m); 7.44 ppm (Ar-H, m); 6.68 ppm (Ar-H, m); 4.60 ppm (CH2, m) and ¹³ C-NMR: -180.0; 150.6; 126.2; 112.7; 159.9; 64.5. The absence of 5-hmf was confirmed by HPLC using an Agilent 1100 system equipped with a 3000 mm BioRad Aminex 87H column using isocratic elution with 25 mM aqueous sulfuric acid at 0.65 ml/min and UV detection. Quantitative determination of 5-hmf was carried out in comparison with an authentic sample. No 5-hmf peak was observed in the oligosaccharide preparation of Example 9, but it was observed after low temperature acid hydrolysis of the oligosaccharide preparation under conditions in which free 5-hmf would not be expected to form in the hydrolysate. Fig. 6 presents an exemplary 2D HSQC spectrum of an oligosaccharide preparation comprising an anti-caramelization subunit.

Example 13: Comparative Example

Commercial 5 kDa dextran was analyzed by MALDI-MS for the presence of anhydrosugar subunits. Fig. 7 illustrates the clear presence of a shifted peak with a -18 g/mol shift from the main DP peak (Na+ adduct at 851.268 g/mol). In contrast, the dextran sample was found to contain virtually no anhydrous sugar subunits.

Example 14 Quantitative determination of anhydro-DP component using LC-MS/MS

The DP2 fraction of the oligosaccharide preparation was isolated by liquid chromatography. Samples were dissolved in water and separated using a Capcell Pak NH2 column (Shiseido; 250 × 4.6 mm, 5 μm) at a flow rate of 1 ml/min under isocratic conditions of water/acetonitrile 35/65. In some cases, 50 μl of 0.05% NH ₄ OH was added after chromatographic separation to enhance ionization. The content of anhydro-DP2 was determined by MS/MS detection. An ESI probe in negative mode was used for MS detection, and the MRM method allowed for targeted analysis. Fig. 8A illustrates the detection of the oligosaccharide drug of Example 9 over the concentration range of 1-80 μg/mL in water with a linear calibration curve (shown in FIG. 8B) versus area under the LCMS/MS chromatogram by concentration.

Fig. 26A-26C, 27A-27C, 28A-28C and 29A-29C illustrate the presence of anhydroDP2, anhydro-DP1 and DP2 species detected by LC-MS/MS in the oligosaccharide preparation of Example 1, Example 3, Example 4 and Example 7, respectively .

Example 15. Preparation of feed containing oligosaccharide preparations

Poultry and pig diets were prepared to demonstrate the inclusion of oligosaccharide drugs in the diet. Control foods showing different ingredient compositions and corresponding treated foods obtained by fortifying the corresponding control foods with the oligosaccharide preparations of Example 9 were prepared as follows.

Sample food 15.1. Control food 15.1 (CTR) was prepared using 62% corn meal and 32% soybean meal. Treated diet 15.1 (TRT) was prepared by adding 500 mg/kg of the oligosaccharide drug from Example 9 to control food 15.1 (CTR). For the treated diet, the oligosaccharide drug was provided in powder form by drying the oligosaccharide on ground rice husks as a carrier and adding the powder into a mixer using a micro-ingredient scale before granulation.

Sample food 15.2. Control food 15.2 (CTR) was formulated using 62% corn meal, 3% soybean concentrate and 26% soybean meal. Treated diet 15.2 (TRT) was prepared by adding 500 mg/kg of the oligosaccharide drug from Example 9 to control food 15.2 (CTR). For the treated diet, the oligosaccharide drug was provided in powder form by drying the oligosaccharide on ground rice husks as a carrier and adding the powder into a mixer using a micro-ingredient scale before granulation.

Sample food 15.3. Control 15.3 (CTR) was formulated using 52% corn meal, 6% corn starch, 5% soybean concentrate, 26% soybean meal and titanium oxide microindicator. Treated feed 15.3 (TRT) was prepared by adding 500 mg/kg of oligosaccharide drug to control feed 15.3 (CTR). For the processed diet, the oligosaccharide drug was provided in powder form by drying the oligosaccharide on crushed rice husk as a carrier and adding the powder to a mixer using a microingredient balance before pelleting.

Sample food 15.4. Control 15.4 (CTR) was prepared using 55% corn meal and 39% soybean meal. Treated feed 15.4 (TRT) was prepared by adding 1000 mg/kg of oligosaccharide drug to control feed 15.4 (CTR). For the processed diet, the oligosaccharide drug was provided in powder form by drying the oligosaccharide on crushed rice husk as a carrier and adding the powder to a mixer using a microingredient balance before pelleting.

Approximate feed 15.5. Control 15.5 (CTR) was formulated using 62% corn meal, 3% soybean concentrate and 26% soybean meal. Treated diet 15.5 (TRT) was prepared by adding 500 mg/kg of the oligosaccharide drug from Example 9 to control food 15.5 (CTR). For the treated diet, the oligosaccharide drug was provided in powder form, with the powder added to a mixer using a weighing scale. for microingredients before granulation.

Sample feed 15.6. Control feed 15.6 (CTR) was a commercial corn soy poultry starter feed from the USA. Treated Feed 15.6 (TRT) was a commercial poultry starter feed made from corn and soybeans in the United States containing 500 ppm of oligosaccharide drug. For the processed diet, the oligosaccharide drug was provided in powder form and the powder was added to a mixer using a microingredient balance before pelleting.

Approximate feed 15.7. Control Feed 15.7 (CTR) was a test corn soy poultry feed with the following diet composition: corn meal 62.39%, soybean meal 31.80%, calcium carbonate 0.15%, dicalcium phosphate 2.2%, sodium chloride 0 .15%, DL-methionine 0.15%, L-lysine 0.10%, soybean oil 2.00%, vitamin-mineral premix 1.00% and coccidiostat 0.06%. Treated feed 15.7 (TRT) was prepared by adding 1000 ppm of the oligosaccharide formulation from Example 9.1 to control feed 15.7 (CTR). For the treated diet, the oligosaccharide drug was provided as a 60% syrup in water and applied by spraying the syrup onto the feed after pelleting.

Approximate feed 15.8. Control Feed 15.8 (CTR) was a test corn soy poultry feed with the following composition: wheat flour 48.45%, soy flour 32.00%, rye 12%, calcium carbonate 0.20%, bicalcium phosphate 2.00% , sodium chloride 0.20%, DLmethionine 0.15%, soybean oil 4.00%, vitamin-mineral premix 1.00%. Treated feed 15.7 (TRT) was prepared by adding 1000 ppm to control feed 15.7 (CTR). oligosaccharide drug from example 9.3. For the treated diet, the oligosaccharide drug was provided as a 60% syrup in water and applied by spraying the syrup onto the feed after pelleting.

As one skilled in the art will appreciate, 15.1-15.6 also contain industry standard levels of fats, vitamins, minerals, amino acids, other micronutrients and feed enzymes. In some cases, the feed contained a coccidiostat, but in all cases it did not contain antibiotic growth promoters. In accordance with standard practice, feed was provided in the form of purees, pellets or crumbs.

Example 16 Extraction of Feed Samples

Diets prepared according to the procedures of Example 15 were processed by extraction. Feed samples were ground in a mill. A sample of five grams of the resulting crushed food was prepared in a 50 ml volumetric flask and hot water (approximately 80°C) was added. After shaking, the mixture was incubated in an ultrasonic water bath at 80°C for 30 min. After cooling, the solution was centrifuged for 20 min at 3000 g, and the supernatant was filtered through a 1.2 μm filter and then a 0.45 μm filter (and in some cases a 0.22 μm filter). The resulting filtered solutions were evaporated to dryness on a rotary evaporator.

In some cases, extraction was performed using 50 wt.% ethanol in water as an alternative extraction solvent. In some cases, the filtration step was performed using a membrane filter with a molecular weight cutoff of 5000 Da.

Example 17 Enzymatic treatment of feed extracts

The feed extracts from Example 16 were subjected to one or more enzymatic hydrolysis steps to break down the oligosaccharides naturally present in the feed. A mixture of α-amylase and amyloglycosidases was used to cleave the α-(1,4) bonds of glucooligosaccharides and starch. Invertase and α-galactosidase were used to remove sucrose, raffinose, and other common fiber saccharides.

Enzyme solutions were prepared as follows: amyloglucosidase (A. niger) 36000 U/g, solution 800 U/ml in ammonium acetate buffer (ammonium acetate 0.2 M, pH 5, containing 0.5 mM MgCl ₂ and 200 mM CaCl ₂ ), α-amylase (from porcine pancreas) 100,000 U/g, Megazyme: solution 800 U/ml in ammonium acetate, invertase from baker's yeast (S. cerevisiae) 300 U/mg, Sigma: solution 600 Units/ml in ammonium acetate buffer; α-Galactosidase from A. Niger, Megazyme 1000 U/ml.

The dry feed extracts from Example 16 were resuspended in 10 ml of ammonium acetate buffer. 50 μl of α-amylase (final concentration 4 U/ml), 50 μl of amyloglucosidase (final concentration 4 U/ml), 50 μl of invertase (final concentration 3 U/ml) were added. If necessary, 20 μl of α-galactosidase was added (final concentration 2 U/ml). The solution was incubated for 4 h at 60°C. The digested extract was then filtered through an ultrafiltration filter (Vivaspin Turbo 4, 5000 MWCO, Sartorius) before evaporation to dryness in a nitrogen evaporation system. In enzymatic digestion embodiments, one or more of the above enzymes were used in combinations and the digestion period varied from 4 hours to overnight digestion. Enzyme concentrations were changed to double the above loadings, and the complete enzymatic digestion procedure was repeated several times in a row on the same feed.

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Table 5. List of feed samples for extraction and digestion

Matrix Solvent Filtration I Enzymatic For split extraction 1 Control food Water 0.22 µM 2 Anhydrooligomeric feed 1000 mg/kg Water 0.22 µM - 3 Control food Ethanol/Water 50/50 0.22 µM - 4 Anhydrooligomeric feed 1000 Ethanol/Water 0.22 µM mg/kg 50/50 5 Anhydrooligomers Water 0.22 µM a + b (4 hours 60°C) 6 Control food Water 0.22 µM a + b (4 hours 60°C) 7 Anhydrooligomeric feed 1000 mg/kg Water 0.22 µM a + b (4 hours 60°C) 10 Control food Ethanol/Water 50/50 0.22 µM - eleven Anhydrooligomeric feed 1000 Ethanol/Water 0.22 µM mg/kg 50/50 12 Anhydrooligomeric feed 1000 mg/kg Water 0.45 µM a + b (4 hours 60°C) 13 Anhydrooligomeric feed 1000 mg/kg Water 0.22 µM a + b (4 hours 60°C) 14 Control starter feed A Water 0.45 µM a + b (4 hours 60°C) 15 Anhydrooligomeric starter feed B 2000 mg/kg Water 0.45 µM a + b (4 hours 60°C) 16 Control food Water 0.45 µM a + b + c (4 hours 60°C) 17 Anhydrooligomeric feed 1000 Water 0.45 µM a + b + c (4 h mg/kg 60°C) 18 Rice and spelled/ Anhydrooligomers Water 0.45 µM 19 Rice and spelled/ Anhydrooligomers Ethanol/Water 50/50 0.45 µM - 20 Control food Water 0.45 µM a + b + c (4 hours 60°C) 21 Anhydrooligomeric feed 1000 Water 0.45 µM a + b + c (4 h mg/kg 60°С) 22 Growth feed C (control) Water 0.45 µM 23 Growth feed D (anhydro-oligomers 2000 mg/kg) Water 0.45 µM 24 Pre-start food A (control) Water 0.45 µM 25 Pre-starter feed D (Anhydrooligomers 1000 mg/kg) Water 0.45 µM 26 Growth feed C (control) Water 0.45 µM a + b + c + (1(4h 60°C) 27 Growth feed D Water 0.45 µM a + b + c + (1(4h (Anhydrooligomers 2000 mg/kg) 60°С) 28 Pre-start food A (control) Water 0.45 µM a + b + c + d(4 ₄ 60°C) 29 Pre-starter feed D Water 0.45 µM a + b + c + d(4 4 (Anhydrooligomers 1000 mg/kg) 60°C) thirty Corn Water 0.45 µM 31 Wheat Water 0.45 µM 32 Soybeans Water 0.45 µM 33 Corn Water 0.45 µM a + b + c + d(4 4 60°C) 34 Wheat Water 0.45 µM a + b + c + d(4 4 60°C) 35 Soybeans Water 0.45 µM a + b + c + d(4 4 60°C) 41 Control food Water 0.45 µM 42 Anhydrooligomeric feed 1000 mg/kg Water 0.45 µM 43 Control food Water ultra 5000 a + b + c X2 (c MWCO overnight at 60°C) 44 Anhydrooligomeric feed 1000 Water ultra 5000 a + b + c X2 (c mg/kg MWCO overnight at 60°C)

Anhydrooligomers refer to oligosaccharides containing anhydrosubunits.

Control foods refer to nutritional compositions without added anhydro-oligomers.

Enzyme a = amyloglucosidase (A. niger) 36000 U/g Megazyme.

Enzyme b = α-amylase (from porcine pancreas) 100,000 U/g Megazyme.

Enzyme c = invertase from baker's yeast (S. cerevisiae) 300 U/mg Sigma.

Enzyme d = α-galactosidase from A. niger 620 U/g Megazyme.

Example 18 Detection of oligosaccharide drugs in feed

Control and treated diets according to Example 15 were analyzed for detection

- 69 044359 absence or presence of oligosaccharide preparations. After production of the feed, 1 kg samples were taken from the final feed. The extractable solids content of the feed was obtained by aqueous extraction using the procedure of Example 16. The resulting extracts were analyzed for the presence of anhydro-DP using LC-MS/MS according to the procedure of Example 14.

Fig. 9 shows the absence of anhydro-DP2 species in the control diet of Examples 15.1 (CTR) to 15.6 (CTR) compared to the presence of anhydro-DP2 species in the treated diet of Examples 15.1 (TRT) - 15.6 (TRT). Integration of the resulting LC-MS/MS chromatograms was used to determine the presence of the oligosaccharide compositions from Example 9 in the final feedstock. Specifically, feeds were determined to contain the appropriate oligosaccharide drug if the area under the anhydrPO-DP2 peak exceeded the detection limit (or any other suitable threshold specified in the method).

Example 19: Quantitative assessment of oligosaccharide preparations in feed

The control and treated diets according to Example 15 were analyzed to determine the concentration of oligosaccharide drugs in the final feed. After production of the feed, 1 kg samples were taken from the final feed. The extractable solids content of the feedstock was obtained by aqueous extraction using the procedure of Example 16. The resulting extracts were analyzed for the presence of anhydro-DP by LC-MS/MS according to the procedure of Example 14, and the peak areas of anhydro-DP2 were compared with standard calibration curve for determining the concentration of the oligosaccharide drug in feed (Table 6).

Table 6. Concentration of oligosaccharide drug in feed

Feed Oligosaccharide content (ppm) in control feed Oligosaccharide content (ppm) in processed feed Example 15.1 Not detected 1642 Example 15.2 Not detected 953 Example 15.3 Not detected 1912 Example 15.4 Not detected 549 Example 15.5 Not detected 406 Example 15.6 Trace amount 401

Example 20. NMR characterization of glucooligosaccharides containing anhydrosubunits

Glucooligosaccharide preparations containing anhydrosubunits were characterized by (i) degree of polymerization and (ii) distribution of glycosidic bonds from glucose units.

Relative molar abundance of α-(1,1)-α, α-(1,1)-β, β-(1,1)-β, α-(1,2), β-(1,2), α (1,3), β-(1,3), α-(1,4), β-(1,4), α-(1,6), and β-(1,6) bonds were identified by NMR - spectroscopy. ¹ H-NMR chemical shifts for anomeric protons were determined as follows: the region d = 4.5-5.5 ppm was considered. with resonances from C-2 to C-6 of covalently bound clusters at ~d 3.2 and 3.9 ppm. Due to the bond with the H atom in C-2, the anomeric proton appears as a doublet, and the axial position appears at a higher field than the equatorial position. The conformation of the sugar was inferred from the binding constant of neighboring protons: equatorial-equatorial, equatorial-axial (small binding constants), or axial-axial (large binding constants).

The identification of glycosidic bonds was also carried out using ¹³ C NMR. Primary, secondary, tertiary and quaternary carbon atoms differ in proton non-resonance decoupling or polarization transfer. Carbons attached to methoxy groups resonate at a lower field than the corresponding carbon atoms with free hydroxy groups, and ring carbon atoms with axial hydroxy groups generally absorb at a higher field than the corresponding carbon atoms with equatorial hydroxy groups. Thus, following these recommendations and comparing with the chemical shifts described in the literature for 1H and ^13C of similar sugars, the assignment of most of the signals was determined.

J-RES and 1H,13C-HSQC were used to determine the binding distribution. For some samples, the HSQC method showed superior performance. For each assay, approximately 50 mg of lyophilized product was dissolved in D2O and transferred to a 5 mm NMR tube. Any residual catalyst or solids were removed by filtration. NMR experiments were performed on a Bruker Avance III NMR spectrometer operating at 600 MHz protons, corresponding to the carbon Larmor frequency of 150 MHz. The instrument was equipped with a cryogenically cooled 5 mm TCI probe. All experiments were performed at 298 K. ¹ H-NMR spectra were recorded and calibrated in deuterated water (4.75 ppm). ¹³ C-NMR spectra were calibrated with acetone (30.9 ppm). Data were acquired using TopSpin 3.5 and processed using ACD/Labs on a personal computer.

Fig. 10 shows a typical 2D-1H JRES NMR spectrum of a sample of gluco-oligosaccharides containing anhydrous subunits, with solvent pre-saturation. The assignments of various glycosidic bonds are made in accordance with the table. 7.

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Table 7. Relative molar abundance of glycosidic bonds in a sample of glucooligosaccharides containing anhydrosubunits (2D-1H JRES NMR method)

Connection Relative molar Relative molar prevalence tested in prevalence tested in laboratories I laboratories II a(1 _? 2) 10.1% 9.2% a(1.4) 2.0% 17.0% a.(1.3) 4.5% 1.3% a(1.6) 28.9% 33.6% β(Ε2) 5.7% 6.5% β(Μ) 13.3% 6.3% β(1,4) 17.9% 10.7% β(1.6) 18.9% 14.5%

As shown in table. 7, for some samples, discrepancies were observed between experiments performed in different laboratories using different 2D-1H JRES NMR analysis instruments.

Fig. 11 shows a typical 1H, ^13C -HSQC NMR spectrum of a sample of gluco-oligosaccharides containing anhydro subunits, with the corresponding resonances and assignments used to assign bonds. In contrast, determination by 1H, ^13C -HSQC NMR appeared to be consistent between two different laboratories and instruments, as shown in Table 1. 8.

Table 8. Relative molar abundance of glycosidic bonds in four samples of gluco-oligosaccharides containing anhydrosubunits (1H, ¹³ C-HSQC NMR method)

Connection Sample 1 Sample 2 Sample 3 Sample 4 Lab I Lab II Lab I Lab II Lab I Lab II Lab I Lab II "(1,2) 9.2% 9.2% 9.0% 9.5% 9.1% 9.9% 9.1% - a(1.4) 1.4% 1.3% 1.2% 1.3% 1.3% 1.3% 0.0% - "PA 17.7% 17.0% 17% 17.7% 17.5% 16.7% 21.9% - a(1.6) 33.9% 33.6% 33.6% 30.9% 36.0% 31.6% 34.4% - β(1Α) 5.7% 6.5% 5.7% 7.6% 5.2% 7.6% 5.2% - β(1,3) 4.1% 6.3% 4.2% 6.2% 4.4% 6.1% 5.6% - β(Μ) 8.5% 10.7% 8.9% 10.7% 7.6% 10.7% 8.3% - β(1Α) 12.3% 14.5% 12.6% 11.6% 11.7% 11.6% 11.0% -

Diffusion-ordered NMR spectroscopy (DOSY) was performed to separate NMR signals of different species according to diffusion coefficient and hence MW. Signals in the upper part of the DOSY spectra in Fig. 12 corresponds to species with high DP value, and species with lower DP value are shown below. Fig. 12 illustrates the overlay of the ^l H DOSY spectra of three oligosaccharides containing anhydrosubunits in table. 8.

Example 21. Semi-preparative isolation of fractions DP1 and DP2

Preparative isolation of the DP1 fraction was carried out by preparative HPLC using a Waters BEH Amide 19x150 mm column. For the mobile phase, water was used as solvent A and acetonitrile as solvent B, where each solvent contained 0.1% ammonia. The applied gradient is shown in table. 9. The collected DP1 fractions from 8 separations were pooled, dried and redissolved in 0.75 ml D2O for NMR analysis as described above.

To characterize the DP2 fraction, a two-step purification was carried out. The first step was carried out in a flash chromatography system using an ELSD (evaporative light scattering detector). 2 ml (2.65 g) of the oligosaccharide preparation was diluted in 1 ml DMSO, 0.5 ml water and 0.5 ml acetonitrile. The solution was stirred and treated with ultrasound for 15 min. 1 ml of solution was injected onto a column YMC DispoPackAT, NH2, spherical beads, 25 μm, 120 g. The oligosaccharide preparation was separated using an isocratic gradient method with 75% acetonitrile in water at a speed of 40 ml/min. The resulting DP2 fraction was dried with nitrogen and redissolved in DMS/water (80:20, v/v).

For step 2 purification, a UHPLC analytical system with a YMC NH2 4.6x250 mm (5 µm) column at 40°C was used. Fraction DP2 was purified using an isocratic gradient (Table 10) and a flow rate of 1 ml/min. A 1:5 split downstream of the column was used to trigger collection by ELSD. Fraction DP2 from 12 chromatography sessions was collected, acetonitrile was removed with heated nitrogen, and residual water was removed by lyophilization. The dry fraction was redissolved for subsequent LCMS/MS and NMR analysis.

Table 9. Gradient method

Time (min) Flow rate(ml/min) Solvent A Solvent B 0 25 10 90 2.5 25 10 90 23 25 25 75 23.1 25 10 90 47 25 10 90

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Table 10. Isocratic method

Time (min) Flow rate(ml/min) Water (%) Acetonitrile (%) 0 1 25 75 15 1 25 75

Example 22. Synthesis of an oligosaccharide drug with a monotonically decreasing DP distribution

330 g of D-glucose monohydrate and 0.3 g of (+)-camphor-10-sulfonic acid were added to a 1 L three-neck flask with mechanical stirring provided by an overhead high-torque mechanical mixer connected through a flexible connector. The flask was secured inside a hemispherical electric heating mantle, controlled by a temperature control device through a thermocouple rod inserted into the reaction mixture. The thermocouple tip was adjusted so that it was in the reaction mixture with a gap of a few mm above the mixing element. The flask was equipped with a reflux condenser cooled by the water-glycol mixture, the temperature of which was maintained below 4°C using a recirculating cooling bath.

The reaction mixture was gradually heated to 130°C with continuous stirring at a stirring speed of 80-100 rpm. When the reaction temperature increased to 120-130°C, the apparatus was switched from a reflux condenser to a distillation configuration with a round bottom receiving flask placed in an ice bath. The reaction was maintained at 130°C with a stirring speed of 120 rpm for 60 min and the mass of condensate collected in the collection flask was recorded over time at 10-min intervals. The reaction was stopped by adding distilled water and stopping heating. After cooling the product mixture to room temperature, an aliquot of the product syrup was diluted to approximately 1° Brix, as determined by refractive index. The diluted aliquot was microfiltered using a 0.2 μm syringe filter and analyzed by HPLC size exclusion chromatography (SEC). SEC analysis was performed on an Agilent 1100 series HPLC with refractive index determination using an Agilent PL aquagel-OH 20 column at 40°C with distilled water as the mobile phase at a flow rate of 0.45 mL/min. MW retention time calibration was performed using standard solutions of known molecular weight. The DP equilibrium constant was determined to be K = 3.3, and the DP distribution was found to decrease monotonically. Fig. 15 and 16 illustrate the DP distribution shape of the various oligosaccharide preparations from Example 9, as determined by HPLC-SEC.

Example 23. Synthesis of an oligosaccharide drug with a non-monotonic DP distribution

330 g of D-glucose monohydrate and 0.3 g of (+)-camphor-10-sulfonic acid were added to a 1 L three-neck flask with mechanical stirring provided by an overhead high-torque mechanical mixer attached through a flexible connector. The flask was secured inside a hemispherical electric heating mantle, controlled by a temperature control device through a thermocouple rod inserted into the reaction mixture. The thermocouple tip was adjusted so that it was in the reaction mixture with a gap of a few mm above the mixing element. The flask was equipped with a reflux condenser cooled by the water-glycol mixture, the temperature of which was maintained below 4°C using a recirculating cooling bath.

The reaction mixture was gradually heated to 135°C with continuous stirring at a stirring speed of 80-100 rpm. When the reaction temperature increased to 130°C, the apparatus was switched from a reflux condenser to a distillation configuration with a round bottom collection flask placed in an ice bath. The reaction was maintained at 135°C with stirring at 120 rpm for 35 min. The reaction was stopped by adding distilled water and stopping heating. After cooling the product mixture to room temperature, an aliquot of the product syrup was diluted to approximately 1°Brix, as determined by the refractive index. The diluted aliquot was microfiltered using a 0.2 μm syringe filter and analyzed by HPLC size exclusion chromatography (SEC). SEC analysis was performed on an Agilent 1100 series HPLC with refractive index determination using an Agilent PL aquagel-OH 20 column at 40°C with distilled water as the mobile phase at a flow rate of 0.45 mL/min. MW retention time calibration was performed using standard solutions of known molecular weight. The DP distribution turned out to be nonmonotonically decreasing. Fig. 16 shows that the content of DP3 is greater than that of DP2, and that the content of DP4 and DP5 is substantially equal.

Example 24. Fed-batch synthesis of an oligosaccharide drug

330 g of D-glucose monohydrate and 0.3 g of 2-pyridine sulfonic acid were added to a liter three-neck flask with mechanical stirring provided by an overhead high-torque mechanical mixer attached through a flexible connector. The flask was secured inside a hemispherical electric heating mantle, controlled by a temperature control device through a thermocouple rod inserted into the reaction mixture. The thermocouple tip was adjusted so that it was in the reaction mixture with a gap of a few mm above the mixing element. The flask was equipped with a reflux condenser cooled by a water-glycol mixture,

- 72 044359 the temperature of which was maintained below 4°C using a recirculating cooling bath.

The reaction mixture was gradually heated to 130°C with continuous stirring at a stirring speed of 80-100 rpm. When the reaction temperature increased to 120-130°C, the apparatus was switched from a reflux condenser to a distillation configuration with a round bottom collection flask placed in an ice bath. The reaction was maintained at 130°C at 120 rpm, and the mass of condensate collected in the receiving flask was recorded over time at 20-minute intervals. After 210 minutes, another 110 g of D-glucose monohydrate was added to the reaction mixture. After 420 min, the reaction was stopped by adding distilled water and stopping heating. After cooling the product mixture to room temperature, an aliquot of the product syrup was diluted to approximately 1° Brix, as determined by refractive index. The diluted aliquot was microfiltered using a 0.2 μm syringe filter and analyzed by HPLC size exclusion chromatography (SEC). SEC analysis was performed on an Agilent 1100 HPLC with refractive index determination using an Agilent PL aquagel-OH 20 column at 40°C with distilled water as the mobile phase at a flow rate of 0.45 mL/min. MW retention time calibration was performed using standard solutions of known molecular weight. The DP equilibrium constant was determined to be K = 0.8, and the DP distribution was found to decrease monotonically.

Example 25 Growth performance of commercial broiler chickens treated with an oligosaccharide preparation

Broiler chickens were raised on the diets from Example 15.6 to determine the effect of the oligosaccharide drug on animal growth performance. Specifically, commercial corn and soybean meal poultry feeds containing a grain fermentation residue solution (DDGS), a coccidiostatic agent, and a standard micronutrient blend were produced according to industry practices and a three-phase feeding program. Through preliminary analysis, feed compositions were determined as shown in Table. eleven.

Table 11. Nutritional Compositions

Component Starter feed Growth feed Weaning food Method Humidity 13.0% 13.0% 12.9% AOAS 930.15 (drawer oven) Crude protein (CP) 24.1% 21.5% 19.6% AOAC 992.15; AOAC 990.03 Fat (EE) 3.2% 3.8% 3.9% AOAC 920.39 (ether extraction) Crude fibers (CF) 2.7% 2.4% 2.4% AOAS 962.09 (hydrolysis) Mineral Residue (AR) 5.2% 4.3% 4.3% AOAS 942.05 (muffle furnace) NFE, by difference 51.9% 55.1% 56.9% Calculated: 1-CP-EECF-AR Total 100.0% 100.0% 100.0%

Control (CTR) and treated (TRT) diets were prepared for each phase as described in Example 15.6, where the treated diets were prepared by fortifying the control diet with one pound per US ton treated using the oligosaccharide preparation from Example 9.7. In total, approximately 50 US tons of each ration were produced.

Hubbard M99 x Cobb 500 chicks were obtained on hatch day from a commercial hatchery and randomly housed in 36 ft x 40 ft pens constructed in a tunnel-ventilated house with a dirt floor. A total of approximately 30,000 birds were housed, with each pen containing the same number of birds. The bedding in the room consisted of loose bedding covered with fresh wood shavings. A standard commercial environmental and lighting program was used. Animals and living quarters were checked daily, including recording of general health status, feed intake, water supply and room temperature. Fatalities were recorded daily.

Birds in odd-numbered pens were fed the treated diet (i.e., containing the feed additive at 2 lb/ton), and birds in even-numbered pens received the control diet. All diets were provided ad libitum through automatic feeders in each pen and feeding trays from days 1 to 7. Water was supplied without restriction from the drinking nipple line.

The start phase ran from day 0 to day 13, the rearing phase from day 14 to day 27, and the stop phase from day 28 to the end of the study, day 31. The weight of the birds in the pen was recorded on days 0 and 31. For each pen, the total weight of the birds consumed was recorded. stern. Weight gain and FCR were then determined for each pen according to standard industry practice.

On day 31, six male birds were randomly selected from each pen for blood and cecal sampling. The live weight of each selected bird was recorded. The blood sample was collected by wing puncture into Vacutainer tubes and frozen after coagulation and serum separation. Each selected bird was then killed by cervical dislocation followed by removal of the cecum using standard veterinary methods. After dissection, the cecal contents were transferred into 15 ml conical tubes, the weight of the cecal contents was recorded, and the contents were snap frozen to -80°C.

Based on the weight of the selected birds, the experimental group showed an increase of 11 points in body weight, significant at P < 0.05 (by ANOVA).

Example 26: Shotgun sequencing of poultry cecal microbiota

The relative abundance of the identified taxa was determined for 96 sampled birds from Study Example 25. For each microbiota sample obtained in Example 25, cecal contents were thawed and DNA extracted using standard methods. Extracted DNA was analyzed on an Illumina HiSeq-X instrument with 2x150 bp reads. Standard analyzes were performed to process the raw sequencing data, including: trimming (adapter, BBDuk), entropy filtering (k = 5, window = 20, min = 50, BBDuk), qualitative filtering (average Q20, BBDuk), filtering Gallus (Bowtie2). Taxonomic assignments were determined from the MetaPhlAn2 database (db_v20).

Example 27: Microbiota Associated with Improved Growth Performance

To assess the phenotypic relationship between microbiota composition and growth performance, sample 25 birds were grouped into cohorts according to their body weight and nutritional efficiency. Contrast analyzes were performed to identify microbiota species that differed significantly between cohorts.

Effect of body weight

First, a correlation analysis was performed by regressing the taxonomic abundance data against the body weight (BW) of the bird from which the sequencing data was obtained. Correlation and correlation P -value were assessed using linear regression to identify potential genera and species that were positively correlated with growth performance measured by body mass. Of the approximately 130 species isolated, fewer than thirty showed a positive BW correlation (Fig. 17).

The selected birds were grouped into two cohorts: (1) Low BW: the lowest quartile of selected birds based on body weight measurements; and (2) high BW: the top quartile of sampled birds based on body mass measurements. Taxonomic data from Example 26 were then compared to identify species with a statistically significant difference in relative abundance between the two cohorts. Specifically, for each OTU value, the relative abundance of the corresponding organism was compared between the two cohorts. Species were identified that showed higher relative abundance in birds with high BW values with P < 0.05, as determined by ANOVA. The cecal microbiota associated with improved BW was characterized by increased relative abundance of the genera Bacteroides, Odoribacter, Oscillibacter, Subdoligranulum, Ruminococcus, and Biophila. The species Odoribacter splanchnicus was found to show a particularly strong correlation with BW. Fig. Figure 17 presents box plots and scatter plots illustrating the four taxa positively associated with BW characteristics (P<0.05).

Impact of Nutritional Efficiency

Correlation analysis was first performed by regressing taxonomic abundance data against the feed conversion ratio (FCR) of the pen corresponding to the bird from which the sequencing data was obtained. Correlation and β-value of correlation were assessed using linear regression to identify potential genera and species positively correlated with growth performance measured by nutritional efficiency. Of the approximately 130 species isolated, fewer than twenty showed a positive FCR correlation.

The selected birds were grouped into two cohorts: (1) high FCR (i.e. low feed efficiency): the top quartile of selected birds as measured by feed conversion ratio; and (2) low FCR (i.e. increased feed efficiency): bottom quartile of selected birds as measured by feed conversion ratio. Taxonomic data from Example 26 were then compared to identify species with a statistically significant difference in relative abundance between the two cohorts. Specifically, for each OTU value, the relative abundance of the corresponding organism was compared between the two cohorts. Species were identified that showed higher relative abundance at low FCR with P < 0.05, as determined by ANOVA. The cecal microbiota, associated with improved feed efficiency, was characterized by increased relative abundance of representatives of the genera Parabacteroides, Akkermansia, Subdoligranulum, Bacteroides, Barnesiella (Fig. 18). The species Bacteroides dorei and Barnesiella gastinihominis showed a particularly strong association with improved feeding efficiency (Figure 18). Fig. Figure 18 presents scatter plots illustrating four taxa positively associated with feeding efficiency (P<0.05).

Example 28. Consistency of taxon associations and inferred function.

Case Study 25 was repeated several times to evaluate the impact of different regions,

- 74 044359 diet types, coccidiosis programs, bird genetics and growth duration. The cecal microbiota obtained by sampling birds and sequencing their gut microbiota according to the methods of Examples 25-25 was repeated for various studies. The contrast analysis and correlation determinations from Example 27 were also repeated for different studies, both independently and after combining the data into a single set.

Taxa that demonstrated consistent associations with efficacy across multiple studies were identified by performing conjoint cohort analyses. In table Figure 12 shows taxa, typical members, and putative function.

Table 12. Microbiota of the cecum

Taxon Typical members Intended function Taxon 1 O. splanchnicus Increased body weight Taxon 2 V. clarus Increased body weight Reduced feed conversion ratio Taxon 3 B. faecalis; V. dorei Reduced feed conversion ratio Taxon 4 B. intestinihominis Reduced feed conversion ratio Taxon 5 Oscillibacter spp. Increased body weight

In addition, over 100 taxa were identified that either showed no correlation with growth characteristics or showed inconsistent associations with growth characteristics between different studies.

Example 29. Method for increasing useful taxa

The effect of oligosaccharide preparations from example 9 on the abundance of beneficial taxa from example 28 was assessed in vivo on commercial broiler chickens. Specifically, the selected birds from the study in Case Study 25 were grouped into two cohorts: (1) a control group consisting of only those birds fed the control diet; and (2) an experimental group, including only those birds that received processed feed containing the oligosaccharide drug.

Contrast analyzes were performed between the control and experimental cohorts. Approximately twenty microbiome species were found to show statistically significant changes in abundance as a result of oligosaccharide feed supplementation. In addition, several taxa associated with the characteristics of examples 27 and 28 were found to increase as a result of feeding the animal an oligosaccharide drug. Specifically: Taxon 4 (Barnesiella intestinihominis) was significantly increased (P<0.01, by ANOVA), and Taxon 1 (Odoribacter splanchnicus) was significantly increased (P<0.1, by ANOVA). Multiple hypothesis testing confirmed that beneficial taxa from Example 29 were significantly increased in the experimental cohort (Q<0.005, Mann-Whitney-Wilcoxon test).

Example 30: Method for Improving Growth Performance

Animals in Study Example 25 fed the treated diets exhibited a statistically significant increase in the number of beneficial taxa and a corresponding statistically significant increase in growth performance, as measured by a treatment effect of 11 points in body weight compared to control (P<0.05).

Example 31 Up-regulation of endogenous digestive enzymes expressed by the gut microbiome

Ex vivo transcriptomics analysis and in vivo feed digestibility studies were performed to demonstrate the ability of the oligosaccharide formulation to increase transcription of genes known to encode enzymes that promote feed digestion and to demonstrate increased feed digestibility in broiler chickens.

Ex vivo transcriptomics

The microbiota sample obtained using the methods of Example 25 was used to prepare a 20% suspension in phosphate buffered saline (PBS) pH 7.4 containing 15% glycerol. Working under anaerobic conditions, an aliquot of the suspension was centrifuged at 2000 g, the supernatant was pipetted, and the pellet was resuspended to form a 1% suspension of the cecal contents in a sterile aqueous mixture: 900 mg/L sodium chloride, 26 mg/L calcium chloride dihydrate, 20 mg /l magnesium chloride hexahydrate, 10 mg/l manganese chloride tetrahydrate, 40 mg/l ammonium sulfate, 4 mg/l iron sulfate heptahydrate, 1 mg/l cobalt chloride hexahydrate, 300 mg/l dibasic potassium phosphate, 1.5 g/ l dibasic sodium phosphate, 5 g/l sodium bicarbonate, 0.125 mg/l biotin, 1 mg/l pyridoxine, 1 mg/l pantothenate, 75 mg/l histidine, 75 mg/l glycine, 75 mg/l tryptophan, 150 mg /l arginine, 150 mg/l methionine, 150 mg/l threonine, 225 mg/l valine, 225 mg/l isoleucine, 300 mg/l leucine, 400 mg/l cysteine and 450 mg/l proline.

Deep well microtiter plates (eg Costar 3958 plates) were loaded in triplicate with 250 μl of the above suspension, 50 μl of a sterile 20% aqueous solution of the selected oligosaccharide preparations from Example 9 and 200 μl of sterile water. Control wells were loaded with 25 μl of a sterile 20% glucose solution and 200 μl of sterile water. The loaded plates were incubated at 37°C for 45 h under anaerobic conditions, followed by RNA extraction with

- 75 044359 using methods known to those skilled in the art. The effect of the oligosaccharide drug on the polysaccharide utilization loci (PULs) of the microbiota was determined by whole transcriptome shotgun sequencing (RNA-seq). Compared with the glucose control, increased transcription of several genes associated with hydrolytic digestion of feed was observed in the presence of the oligosaccharide drug: GH127 (CAZyme a-L-arabinofuranosidase), susC (outer membrane receptor associated with the starch utilization system) and susD (starch binding protein). Fig. 19 illustrates increased transcription of hydrolytic digestive enzymes belonging to PUL in microflora treated with an oligosaccharide drug compared to controls.

Increasing feed digestibility in vivo

Ross 308 broilers were raised for 35 days in raised-floor pens on a traditional three-phase feeding program. Three dietary treatment groups were compared: (1) a negative control group fed a typical industry wheat-soybean poultry diet; (2) a positive control group fed a control diet supplemented with a conventional xylanase feed additive (AB-Vista Econase) at the recommended inclusion level; and (3) a test group fed a control diet supplemented with 500 ppm of the oligosaccharide formulation of Example 9. All diets contained coccidiostats, industry standard levels of phytase and the TiO ₂ marker to perform digestibility determinations. Standard environmental, lighting and monitoring programs were used. A randomized block study design was implemented with eight pens per treatment group with thirty-eight birds per pen.

Birds in the experimental group showed a statistically significant increase in dry matter digestibility by 2.8% and a statistically significant increase in nitrogen digestibility by 4.5% compared to the negative control group. The experimental group also outperformed the positive control group fed the enzyme-supplemented diet. Birds from the positive control group showed an increase in dry matter digestibility by 1.4% and nitrogen digestibility by 1.5%.

Example 32. Raising and sampling of piglets treated with an oligosaccharide preparation

Weaned piglets were raised on the diets of Example 15 to determine the effect of the oligosaccharide formulation on the functional metagenomics of porcine ileal, cecal, and fecal microbiomes.

One hundred and forty-four Redon x Large-White weaned pigs with an initial body weight of 8.54 ± 1.70 kg were reared for 42 days in a single-deck battery cage in an environmentally controlled facility. The animals were divided into 4 equal groups of 36 piglets in 9 pens with 4 animals per pen. Each pen had a plastic-coated welded wire bottom and two nipple drinkers and two individual stainless steel feeders. Room humidity was maintained at 50% and temperature was initially maintained at 27°C and decreased weekly by approximately 2°C per week to 21–22°C. The animals were fed in accordance with a two-phase dietary program compiled in accordance with the nutritional recommendations of the NRC (2012) using the diet composition given in Table. 13.

Table 13. Composition of diets for weaned piglets

Ingredient Pre-start feed (wt.%) Starter feed (wt.%) Corn 52.20 62.25 Soy flour 26.00 26.00 Corn starch 6.00 - Soy protein concentrate 5.00 3.00 Calcium carbonate 0.60 0.55 Dicalcium phosphate 1.7 1.50 Sodium bicarbonate 0.45 0.40 Sodium chloride 0.25 0.20 L-lysine 0.80 0.65 L-threonine 0.30 0.25 L-methionine 0.20 0.20 Soybean oil 3.00 2.00 Vitamin and mineral premix 3.50 3.00

The pre-starter diet was given from days 0 to 14, and the starter diet was given from days 15 to 42. All rations were given without restriction in the form of mash. The piglets were divided into experimental groups in accordance with table. 14.

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Table 14. Experimental groups of weaned piglets

Experimental group Pre-start diet Starter diet Control Ration 15.3 (CTR) Example 15.5 (CTR) Experimental group A Diet 15.3 (CTR) supplemented with 125 ppm oligosaccharide preparation 9.7 Diet 15.5 (CTR) supplemented with 125 ppm oligosaccharide preparation 9.7 Experimental group B Diet 15.3 (CTR) supplemented with 250 ppm oligosaccharide preparation 9.7 Diet 15.5 (CTR) supplemented with 250 ppm oligosaccharide preparation 9.7 Treatment Diet 15.3 (TRT) Diet 15.6 (TRT)

The average daily weight gain (ADWG) of piglets in the experimental group was determined to be 508.1 g/day compared to the control group, whose weight gain was 494.9 g/day. The feed conversion ratio for the control group was 1.781 kg/kg, and for the experimental group - 1.748.

Example 33. Effect of an oligosaccharide drug on functional metagenomics

Weaned piglets were raised in pens according to the general protocol from Example 32. The piglets were divided into two experimental groups: the control group received diets 15.3 (CTR) and 15.5 (CTR), and the experimental group received diets 15.3 (CTR) and 15.5 (TRT ). For each experimental group, 13 animals were randomly selected, sacrificed, and dissected to obtain ileal, cecal, and fecal (colon) microbiota samples.

microbiota samples were prepared by SAMBO DNA extraction and sequenced using MetaQuant (MGP). On average, 21.6 million cleaned reads were obtained per sample. Mapping and counting were performed using METEOR. Mapping with Bowtie2 (95% identity) was performed to remove host contaminants (pig genome) and the resulting sequences were profiled against a custom gene catalog containing 9M genes (ileal, cecal and fecal microbiomes). Biostatistical analysis was performed using MetaOMineR. More than 2 x 10 ⁷ high quality reads were obtained. To avoid bias due to mapping rate variability, all samples were reduced to 10M mapped reads and gene abundance was normalized using FPKM according to gene length and sequencing depth.

Gene richness and functional metagenomic analysis

Samples were grouped into cohorts according to experimental group and microbiome type (ileum, cecum, feces), and contrast analyzes were performed to determine statistically significant changes in gene richness, measured as the number of individual genes. Animals in the experimental group showed a 2-5-fold increase in gene richness, significant at P<0.006 for cecal microbiota and P<0.06 for fecal microbiota (Wilcoxon test). There was also a numerical increase in ileal gene richness.

Genes were annotated using the KEGG database (v82) and grouped according to gut metabolic modules (GMM) [Darzi, Y. et al., The ISME Journal, 10, 1025-1028 (2015)] using an internal line. Contrast analysis was performed on GMM prevalence data to determine the metabolic pathways and functions that were modulated in the experimental group compared to the control group. Fig. 20 illustrates the effect of an oligosaccharide drug on the functional metagenomics of the piglet microbiome. A statistically significant increase in pathways associated with acetogenesis was observed in the ileal microflora. Statistically significant increases in pathways related to pectin degradation and SCFA production, among others, were observed in the cecum. In addition, SCFA pathways for butyrate and propionate production were enriched in the cecal microbiota for the experimental group.

Example 34. Reduction of undesirable species of microorganisms in the intestinal microflora

The effects of the oligosaccharide preparations of Example 9 on undesirable taxa were assessed in vivo in commercial broiler chickens. Specifically, the selected birds from the study in Case Study 25 were grouped into two cohorts: (1) a control group consisting of only those birds fed the control diet; and (2) an experimental group, including only those birds that received processed feed containing the oligosaccharide drug.

Contrast analyzes were performed between the control and treatment cohorts to assess changes in the abundance of the taxa analyzed in Example 26. Birds fed the treated feed exhibited the following changes in the relative abundance of microorganisms, as shown in Table 1. 15.

Table 15. Relative number of microorganism species in the experimental group

Types of microorganisms logz (fold change compared to control group) Campylobacter jejuni -1.9 (P < 0.001) Helicobacter pullorum -1.3 (P<0.001) Campylobacter coli -0.7 (P < 0.05) Escherichia coli -0.7 (P<0.1)

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Fig. 21 shows that the types in table. 15 decreased in the experimental group compared to the control group. In contrast, 83 species either increased or did not decrease in relative abundance.

Experiments on growing one strain confirmed that oligosaccharides do not have an antimicrobial effect.

Example 35: Consistency across multiple studies

The study from Example 25 was repeated several times to evaluate the effects of different regions, diet types, coccidiosis programs, bird genetics, and growth duration. Cecal microbiota obtained from avian samples in additional studies were sequenced according to the methods of Examples 25-26. The contrast analysis from Example 34 was also repeated for different studies, both independently and after combining the data into a single set. Reducing the number of gram-negative bacteria, Campylobacter spp., Heliobacteria spp. and Escherichia spp. has been consistently observed in several studies. Consistency in Proteobacteria Campylobacter species was observed across all studies, as was an overall decrease.

Example 36: Relationship between Mortality and Enteric Pathogen Abundance

The effects of common gut microflora pathogens on animal health were assessed in broiler chickens.

Unsexed Ross 708 chicks were obtained from conventional breeders and housed in twenty-four 8-foot by 6-foot floor pens located within a single broiler house. Thirty male chicks were housed per pen, and the weight of birds in the pen was recorded on day 0. Birds were raised on litter amended with fresh wood shavings to provide approximately 4 inches of litter. The room temperature was monitored and recorded daily. Environmental conditions during testing were consistent with industry practice. All birds were vaccinated according to the standard vaccination program and were additionally vaccinated against coccidiosis.

Birds were fed diets in two experimental groups in accordance with a four-phase feeding program, as shown in Table. 16.

Table 16. Four-phase feeding program

Diet phase Feed type Age (days) Starting Chit 0-16 Rostovaya Small granules 16-28 Finish Granules 28-42 Withdrawal Granules 42-49

All diets were corn-soybean meal based and contained dry grain fermentation residue (DDGS) and were free of growth promoting antibiotics and ionophores or other coccidiostats. Birds in the control group (CTR) were fed the basal diet, while birds in the test group (TRT) were fed the basal diet supplemented with 500 ppm of the oligosaccharide preparation from Example 9.

The diet was provided ad libitum through commercial Choretime automatic feeders in each pen. From days 1 to 7, food was also served on feeders placed on the bedding. Water was supplied without restriction from one nipple drinking line. Standard floor pen management practices were used throughout the experiment. Animals and living quarters were checked twice daily, observing and recording general health, constant feeding and water supply, and temperature, removing any dead birds, and recognizing unexpected events. Pen number and date of removal were recorded for all dead and slaughtered birds during the study.

On day 15, one bird randomly selected from each pen had cecal fluid and microbiota sampled following the procedure in Example 25, and the microbiota was analyzed according to the methods in Example 26. The selected birds were grouped into two cohorts: (1) birds with birds taken from pens in the top quartile of mortality had high mortality; and (2) birds with low mortality were birds taken from pens in the lowest quartile of mortality. Contrast analysis was performed to identify microbial species with statistically significant differences in abundance between the two cohorts. About five species were found to be highly correlated with mortality. Little or no correlation was found for over a hundred other taxa of the cecal microbiota. A particularly strong correlation was observed between the relative abundance of Escherichia species and mortality. Fit. Figure 22 presents a scatterplot for Escherichia coli (left panel) and other Escherichia species (right panel), clearly illustrating the relationship between pathogen abundance and bird mortality.

Example 37. Therapeutic effect of oligosaccharide drugs

Selected birds from example 36 were analyzed into experimental groups, for which a threefold decrease in the relative abundance of Escherichia coli was observed. Overall, the number of Escherichia species decreased (Q = 0.07, multiple comparison test by Mann-Whitney-Wilcoxon test). In addition, the selected birds in the experimental group showed a threefold decrease in the number of pens in which the apicomplexan parasite Eimeria tenella was detected.

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Example 38: Meta-analysis of studies on live growth performance of broiler chickens

The effects of oligosaccharide formulations on live growth performance of commercial broiler chickens were assessed in vivo through a series of independent studies conducted across different regions, seasons, basal diet types, avian genetics and management practices, including litter treatments and coccidiosis control programs. In each study, birds were assigned to experimental groups, including one control group and one or more experimental groups. The control group was fed only the background diet. The experimental groups were given a background diet supplemented with a certain dose of oligosaccharide preparations from Example 9. In some studies, a commercial feed additive used in poultry farming was included as a comparative example.

For each study, birds were housed in pens located in a typical broiler house with a specified number (Hd/Rep) of birds in each pen. Statistical replicates were performed by randomly assigning pens to experimental groups with a specified number (Reps/Trt) of replicates per experiment. In table Table 17 provides detailed protocol information for each study included in the analysis.

Table 17. Protocols

Study A country Season Duration Type of diet Replication/experiment Quantity/repetition Genetics Floor Litter Program against coccidiosis Etc. 38.1 USA Spring 35 Corn/Soybeans 6 14 Cobb 500 M/F Use Saccox Etc. 38.2 USA Winter 49 Corn/Soybeans 12 60 Cobb 500 M/F Use Maxiban Etc. 38.3 Canada Winter 35 Corn/Soybeans 8 60 Ross 708 M Use Saccox Etc. 38.4 USA Winter 49 Corn/Soybeans 12 60 Cobb 500 M/F Use Maxiban Etc. 38.5 Conn. Kingdom n/a n/a Wheat/Soybean n/a n/a Η/Π n/a Clean No Etc. 38.6 USA Winter 33 Corn/Soybeans 12 100 Cobb 500 M/F Use Amprol Etc. 38.7 USA Summer 49 Corn/Soybeans 12 18 Hubbard M99 M Use No Etc. 38.8 Canada Winter 42 Corn/Soybeans 12 17 Ross308 M Use Vaccine Etc. 38.9 Great Britain Autumn 42 Wheat/Soybean 16 35 Ross308 M Fresh No Etc. 38.10 France Autumn 42 Wheat/Soybean 17 thirty Ross308 M Fresh No Etc. 38.11 USA Autumn 42 Corn/Soybeans 21 40 Cobb500 M Use Vaccine Etc. 38.12 France Summer 36 Corn/Soybeans 12 18 Cobb500 M Fresh Vaccine Etc. 38.13 Canada Spring 42 Corn/Soybeans 10 20 Ross708 M Use Vaccine Etc. 38.14 USA Spring 42 Corn/Soybeans 14 40 Cobb500 M Use Vaccine Etc. 38.15 New Zealand Spring 35 Wheat/Soybean 12 20 Ross308 M Fresh Vaccine

Study results included bird weight (BW), feed intake (FI), feed conversion ratio (FCR), mortality rate (per head) and mortality rate. The pen was a statistical unit. Spatial blocking was used whenever possible, and experimental groups were randomly assigned to blocks.

Background rations

Birds were fed phased diets according to local commercial practices for a total study duration of 35 to 49 days. Starter phase diets were typically provided in kibble form from the time birds were housed until day 15 of the study. All diets did not contain growth promoting antibiotics. The designs of the starter control diet are described in Table. 18 (NA = data not available from the site).

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Table 18. Compositions of starter control diets

Study Corn flour, % Wheat flour, % Soy flour, % Fermentation residue solution corn,% | Unpurified protein Unrefined fat True metabolic value (kcal/kg) Lysine (SID) Methionine (SID) Etc. 38.1 63.5 n/a 27.4 n/a 22.1 N/A 2988 1.35 N/A Etc. 38.2 0 n/a 0 n/a N/A N/A N/A N/A n/a Etc. 38.3 63.4 n/a 28.3 n/a 20.9 N/A 2940 1D4 n/a Etc. 38.4 N/A n/a n/a n/a n/a N/A N/A N/A n/a Etc. 38.5 n/a n/a n/a n/a n/a n/a N/A N/A n/a Etc. 38.6 0 n/a 0 n/a N/A n/a ZON N/A n/a Etc. 38.7 0 n/a 0 n/a n/a n/a N/A N/A n/a Etc. 38.8 54.52 0 34.38 5 22.23 2.81 2900 1.24 0.63 Etc. 38.9 0 51.78 30.5 0 21.31 5.74 2899 1.251 0.622 Etc. 38.10 0 55.1 28 0 22.49 5.42 2899 1.237 n/a Etc. 38.11 58,353 2,377 29,992 5 20.3 N/A 2900 1.33 N/A Etc. 38.12 N/A N/A N/A N/A 22 N/A ZON n/a n/a Etc. 38.13 56.08 0 34.1 5 22.23 2.31 2900 1.24 0.63 Etc. 38.14 58,353 0 29,992 5 20.3 N/A 2900 1.33 n/a Etc. 38.15 0 54.92 28.31 5 n/a 6.9 2900 1.24 n/a

Growth phase diets were provided in pellet form from days 16 to 24. The compositions of control growth diets are described in detail in Table. 19 (N/A = data not available from the site).

Table 19. Compositions of control diets for the growth phase

© S X Λ X ! ς CJ CJ s Project 38D Corn flour, % I Wheat flour, % 22 Soy flour, %, sch Solution of the residue after corn fermentation, % Unpurified protein I'm unrefined fat g True ίο metabolic ► value (kcal/kg) Lysine (SID) n/a Θ X X X © X δ Ex.38.2 N/A N/A N/A N/A N/A N/A N/A N/A n/a Project 38.3 65.6 N/A 26.3 N/A 19.9 N/A 2988 1.06 n/a Project 38.4 N/A n/a N/A N/A N/A n/a N/A n/a n/a Project 38.5 N/A n/a n/a N/A N/A n/a N/A n/a n/a Project 38.6 N/A n/a n/a N/A N/A n/a 3102 n/a n/a Project 38.7 N/A n/a n/a N/A N/A n/a N/A n/a n/a Project 38.8 54.95 0 28.31 10 20.8 3.88 3000 1.11 0.56 Project 38.9 0 57.135 26 0 19.01 6.55 2997 1.08 0.533 Project 38.10 0 55.77 24 0 20.94 7.4 2998 1.11 n/a Project 38D1 65,383 0.344 20,404 10 17.5 N/A 3040 1.33 n/a Project 38.12 N/A N/A N/A N/A 19 N/A 3035 n/a n/a Project 38.13 56.53 0 28.02 10 20.8 3.39 3000 1.11 0.56 Project 38.14 65,383 0 20,404 5 17.5 N/A 3040 1.14 n/a Project 38.15 0 57.3 23.05 6 N/A 6.27 3000 1.11 n/a

Terminal phase diets were provided in pellet form from days 16 to 24. The compositions of control diets of the final phase are described in detail in Table. 20 (N/A = data not available from the site).

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Table 20. Compositions of final phase control diets

Study Corn flour, % Wheat flour, % Soy flour, % Residue solution after corn fermentation, % Unpurified protein Unrefined fat True Metabolic Value (kcal/kg) Lysine (SID) Methionine (SID) Etc. 38.1 74.3 N/A 27.4 N/A N/A N/A 3155 1.06 N/A Etc. 38.2 N/A N/A N/A n/a n/a N/A N/A n/a n/a Etc. 38.3 70.8 n/a 28.3 n/a n/a n/a 3059 0.94 n/a Etc. 38.4 N/A n/a N/A n/a n/a n/a N/A N/A n/a Etc. 38.5 n/a n/a n/a n/a n/a n/a N/A N/A n/a Etc. 38.6 n/a n/a n/a n/a n/a n/a 3203 N/A n/a Etc. 38.7 n/a n/a n/a n/a n/a n/a N/A N/A n/a Etc. 38.8 57.37 0 24.85 10 19.4 5.16 3100 1.03 0.55 Etc. 38.9 0 59.94 23 0 17.53 7.71 3097 1.003 0.503 Etc. 38.10 0 87.67 21 0 19.53 8.88 3099 0.994 N/A Etc. 38.11 69.41 0.12 16,879 10 16 N/A 3084 1.01 N/A Etc. 38.12 N/A N/A N/A n/a N/A N/A N/A N/A N/A Etc. 38.13 58.95 0 24.56 10 19.4 4.66 3100 1.03 0.55 Etc. 38.14 69.41 0 16,879 5 16 N/A 3084 1.01 N/A Etc. 38.15 0 62.02 17.62 6 N/A 7.25 3100 0.99 N/A

Experimental groups

For each study, experimental groups were created to compare the effect of oligosaccharide drugs with a control diet. For selected studies, experimental groups were designed to evaluate the dose-response curve for oligosaccharide drugs. Treated diets were prepared by mixing a sufficient amount of the appropriate oligosaccharide preparation from Example 9 such that the final oligosaccharide content reached the target dose (units ppm on a dry solids basis). In separate studies, a comparative example (Comparative Example 36) was provided by a commercial whole yeast product (Diamond V XPC Original). The experiment was carried out in accordance with table. 21.

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Table 21. Distribution of experimental groups

Study Experimental group 1 Experimental group 2 Experimental group 3 Experimental group 4 Experimental group 5 Experimental group 6 Experimental group 7 Etc. 38.1 Control Etc. 9.7 (500 ppm) Etc. 38.2 Control Etc. 9.7 (500 ppm) Etc. 38.3 Control Etc. 9.7 (500 ppm) Etc. 38.4 Control Etc. 9.7 (500 ppm) Etc. 38.5 Control Etc. 9.7 (100 ppm) Etc. 9.7 (500 ppm) Comp. Project 36 (1500 ppm) Etc. 38.6 Control Etc. 9.7 (500 ppm) Etc. 38.7 Control Etc. 9.7 (500 ppm) Etc. 38.8 Control Etc. 9.7 (500 ppm) Etc. 9.3 (500 ppm) Etc. 38.9 Control Etc. 9.7 (500 ppm) Etc. 38.10 Control Etc. 9.7 (500 ppm) Etc. 9.2 (500 ppm) Etc. 9.3 (500 ppm) Comp. Etc. 36 (1250 ppm) Etc. 38.11 Control Etc. 9.7 (500 ppm) Etc. 9.2 (500 ppm) Etc. 9.3 (500 ppm) Etc. 9.4 (500 ppm) Etc. 9.5 (500 ppm) Comp. Etc. 36 (1250 ppm) Etc. 38.12 Control Etc. 9.7 (500 ppm) Etc. 9.3 (500 ppm) Etc. 38.13 Control Etc. 9.2 (100 ppm) Etc. 9.2 (250 ppm) Etc. 9.2 (500 ppm) Etc. 9.2 (750 ppm) Etc. 9.2 (1000 ppm) Etc. 38.14 Control Etc. 9.3 (100 ppm) Etc. 9.3 (250 ppm) Etc. 9.3 (500 ppm) Etc. 9.3 (750 ppm) Etc. 9.3 (1000 ppm) Etc. 9.7 (500 ppm) (continued, 8-13) Etc. 9.2 (100 ppm) Etc. 9.2 (250 ppm) Etc. 9.2 (500 ppm) Etc. 9.2 (750 ppm) Etc. 9.2 (1000 ppm) Comp. Etc. 36 (1250 ppm) Etc. 38.15 Control Etc. 9.2 (100 ppm) Etc. 9.2 (250 ppm) Etc. 9.2 (500 ppm) Etc. 9.3 (100 ppm) Etc. 9.3 (250 ppm) Etc. 9.3 (500 ppm)

Standard equipment and methods known in the art were used to prepare both the background and treated diets. For the treated diets, oligosaccharide preparations and comparison products were prepared on top of the background diet and added to the pre-pellet mixer. Inclusion of oligosaccharides was confirmed by feeding assay.

Growth phase and sampling

Food and water were provided without restrictions. Commercial lighting and temperature programs were implemented in each study in accordance with local industry practices in the respective region. Pens were checked daily and the number and percentage of any deaths recorded in the study log. No veterinary intervention was required.

For each diet phase, total pen weight gain, initial and final bird numbers, and total feed intake for each pen were measured. For each pen, average bird weight (BW) was calculated by dividing the total weight in the pen by the number of birds in the pen at the time of weighing. For each paddock, feed conversion ratio (FCR) was calculated by dividing the total feed intake for the interval by the total weight gain of the corresponding paddock. FCRs were adjusted for mortality (FCRma) by adding the overall percentage of deaths for the period. To account for differences in feather weight, FCRma was adjusted for total body weight to obtain a adjusted FCR (cFCR) for each pen using methods known in the art. The correction factor was determined for different avian genetics using published growth day-specific BW and FCR targets for the respective genetics.

In the selected studies, one bird was randomly selected from each pen for sampling on either day 15 or the last day of the study. For each sampled bird, 5 ml of blood was collected from a wing vein into vacuum serum containers. After coagulation, the serum was separated by centrifugation, recovered, and frozen on dry ice for later processing. Each selected bird was then euthanized according to local ethical procedures and necropsied. Cecal contents were collected into 5 ml conical tubes and immediately frozen for whole genome microbiome sequencing and cecal metabolomics. Minor ileal tissue resection was performed, and the tissue was processed to deactivate RNA and frozen for subsequent gene expression analysis.

Example 39. Meta-analysis of a study

A statistical meta-analysis of the in vivo studies from Example 38 was performed to evaluate the effects of oligosaccharide feed additives and comparator products on poultry performance compared to birds fed control diets. The analysis used a linear mixed model with treatment group as a fixed effect and random effects for study nested within block. Statistical analysis was performed in R version 3.4.4 (2018-03-15). The results were assessed using the least squares method with statistical significance P<0.05. Pairwise comparisons were performed using Tukey's method, assigning letter designations: a, b, c, d,... Treatments without a common letter in their Tukey grouping were significantly different in pairwise comparisons at P < 0.05.

Feed Conversion Ratio

Study effects for cFCR were significant at P < 0.05. The oligosaccharide treatment provided a cFCR improvement of at least 2.7% at 500 ppm versus the control diet, compared to the comparative example which provided a 2.2 unit improvement in cFCR at 1250 ppm. The oligosaccharide from Example 9.4 provided a 6.4 unit improvement in cFCR when included at 500 ppm. The meta-analysis results for cFCR are presented in Table. 22.

Table 22. Meta-analysis of cFCR

Experimental group cFCR (Is average) S.E. df Tukey grouping D cFCR (compared to control) Control 1.6511 0.043 14 d Etc. 9.2 (500 ppm) 1.6019 0.044 14 A -4.9 Etc. 9.3 (500 ppm) 1.6106 0.044 14 abc -4D Etc. 9.4 (500 ppm) 1.5876 0.044 14 A -6.4 Etc. 9.5 (500 ppm) 1.5948 0.044 14 ab -5.6 Etc. 9.7 (500 ppm) 1.6246 0.043 14 bc -2.7 Comp. Etc. 36 (1250 ppm) 1.6293 0.044 14 With -2.2

The oligosaccharide groups showed greater consistency of effect compared to Comparative Example 38. For each oligosaccharide included across multiple studies, the consistency of its effect on cFCR was assessed by determining the proportion of studies that observed a given amount of improvement in cFCR compared to control. For example, the oligosaccharide from Example 9.2, when included at 500 ppm, provided at least 3 units of cFCR improvement in 80% of studies, at least 4 units of cFCR improvement in 60% of studies, at least 5 units of cFCR improvement in 40% of studies, and at least 6 units of cFCR improvement in 40% of studies. The comparator at inclusion of 1250 ppm provided a cFCR improvement of 3 units in only 25% of the studies and did not provide a cFCR improvement of 4 units or greater in any of the studies. The results are presented in table. 23.

Table 23. Consistency of application effect on cFCR

Experimental group 1 improvement unit 2 upgrade units 3 upgrade units 4 improvement units 5 upgrade units 6 upgrade units Etc. 9.2 (500 ppm) 100% 80% 80% 60% 40% 40% Etc. 9.3 (500 ppm) 100% 83% 67% 67% 50% 33% Etc. 9.7 (500 ppm) 85% 85% 38% 23% 15% 0% Comp. Etc. 36 (1250 ppm) 100% 100% 25% 0% 0% 0%

A clear dose-dependent relationship was observed between cFCR and the degree of oligosaccharide inclusion in the diet. For the oligosaccharide from Example 9.2, a 2.4 unit improvement in cFCR was observed with 100 ppm inclusion (P>0.05), a 3.7 unit improvement in cFCR was observed with 250 ppm inclusion (P<0.05) , a 6.4 unit improvement in cFCR was observed with 1000 ppm inclusion (P<0.05).

Bird weight

Study effects for BW were significant at P < 0.05. The oligosaccharides provided an increase in body weight of at least 48.9 g compared to the control diet at 500 ppm, compared to the comparative example, which provided a 39.6 g increase in body weight at 1250 ppm. . The oligosaccharide from Example 9.5 provided an increase in body weight of 81.8 g compared to the control when included at 500 ppm. The results of the meta-analysis of poultry weight are presented in Table. 24.

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Table 24. Meta-analysis of bird weight

Experimental group BW (Ismean) S.E. df Tukey Grouping ABW (g vs control) Control 2.755 FROM 14 A 0 Etc. 9.2 (500 ppm) 2.817 FROM 14 b 62.6 Etc. 9.3 (500 ppm) 2,807 from 14 b 52.6 Etc. 9.4 (500 ppm) 2.838 114 14 b 83.3 Etc. 9.5 (500 ppm) 2.837 114 14 b 81.8 Etc. 9.7 (500 ppm) 2,804 FROM 14 b 48.9 Comp. Etc. 36 (1250 ppm) 2,794 FROM 14 b 39.6

Group homogeneity

Birds fed the 500 ppm oligosaccharide formulations showed improved group uniformity compared to birds fed the control diet. For each treatment group, group homogeneity was assessed by calculating the proportion of bird weight that was within ±5% of the mean bird weight for the corresponding study. On average across all studies 36.1-36.15, 81.7% of birds fed the control diet weighed within ±5% of the average bird weight, while 91.3% of birds fed the oligosaccharide-supplemented diets from Example 9.2 is within ± 5% of the average bird weight. The effect of homogeneity was significant at P < 0.01 as measured by the nonparametric Ansari-Bradley test.

Example 40: Microbiome Sequencing and Correlation Analysis

Cecal microbiome samples from Example 38 were processed and analyzed by whole genome sequencing using methods comparable to those described in Example 26. The relative abundance of identified taxa was determined and correlated with poultry performance parameters including feed efficiency ratio (FCR) and body weight (BW), by linear regression. As shown in table. 25, the following taxonomic lineages provided the 94 strongest correlations with performance endpoints.

Table 25. Taxonomic lineages with the 94 strongest correlations with endpoints

Corre- & _W e- Taxonomic line lshtsh -TNTPG5G B.W. FCR k._Bmnenxp_Laih^lwrac:c_Un|i!^kTcnw ssifiid.g_VktnaltalesjuKlaisiritd:s_Vi€[naM^^ 0 127 M2 P till . . Λ kBactcnaip. Gammaprotcobactenaro F.nrcrobacteraleM', Entero 0 pchk bts1paazh;v Escherichia^ Escherichia fcrgn.wnii W G kZ3 ^aCKO 'Tp_Fusobactcria:c_Fii5obacKriia:o_Fu^iacteriaks.f_Fu^^^ 0 schch 0 274 e__Fu5obactcnum.5_FiisobacU'niini_sp_CAG_41 ^< ' '54 A2 k_Vas1epa:p__Fmnicutcs.c_Ncgati\ iciitesiu_Sekinjmonadalcs.%SdenomonadaLVa OPKV 0 24/ e.g__McganJyiiasHhiditeSilkd IS

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t__Bacteria:p_BacrciOidetes,e__Bacteroidia.o__Bactei'£jidak4.,f__OdoribactoraceaciUii ciass i f to d. U nc 1 assi il cd UA37 U7 9210 X5 k_ Bnclcria:p Firmictitosx . En sipdotrichin.n Ensipdotriclialcs.f Eiysipclotncha ceac; Un cl assi find; Unci assi tkd ().075 97 0 200 7ft k__Ba«cria.p__Fiimicutcsx__Ctostridia.o__Cbstndiak’sT__Rummococcaceac.g__Fae cattbacknum.s__Faucalibacteriumjjrausnitzii 0.066 03 o |97 98 k, Bactcriatp. Firmicutex Clnstricha;n Clnstndiafcs:f Clastriihacuac'.g Candida Ins Arthromitus;? Candidates Arthmmitus sp SFB luAcy 0.209 42 0 ШЧ7 39 k__Bactciia.p__Firmicirtes.c__Clostridia.o__Cbstndialesif__LacliituspiraL-eac.g__Lach rioclostiidiutnis__LadHioclosttidiuiii_sp_Anl4 0455 fk 11 196 u k_Bacteria;p___Racwroidctesx_Bacteroidia.c,_BactcroiddcsJ<bcfcroida^^ _B actcroidcs'5 Bactcmidcs intcstinalis 0.141 503 0 N5 81 k__Buclcnaip__Fmnieirtes.c_Ck)sindixc><dostndialevf_Ladmiwpiuic^ isockistridium.s__Ladwocl0snidiuin_sp_An 138 0071 (. « 0 1Ш 79 k _ BactcrLxp Proton bacteria;c Dcltaprotoobactena.o DcsulfovibrionalcsT Desulf OMbi'innaccac;u__Riloplii la. Unclassified 0 252 334 0 IKA 3' k__BaCtorta;p_Proto4)baLk'riu.c__DdUprMeilhil£tcm.u__Dcsuifnribrinnalcs;f_Dcsulf onbnoriaLTae;g_Dcsulfonbnonac^jindi^fiL4l:s_DesuifoMbrionac^^ 0.170 Uh 0 IS2 4 k, Bactoria;p . F!miiciitosto_.Ciostndia;Q ,Chstndfaks:f. Kshitsossaseas^ . Ana cromincus.s, Anacrotnineus colihoi^ 04 17 15 π 183 k__Bactoria:p._Firmicutos;c__Ckistridiu:o _ Clt>s1nduil^:f_Ruminoc0c^^ eromassdibacillusis__Aiuicrumassilibacdlus_sp_Anl72 O.OhO 701 0.180 53 k_Bacteria^. Protoobacteria:c_Betaprotoobactcria:o .Vigk1yu14spa1s54'_ BniLholdc rialcs_unclassiflcd.ii Buddwldcriales tmchssificds _Burkholdcriafe bacterium 1J 47'' U.079 419 0 ISO eleven k_ Bactena;p Hrmicutosx _ Erysipdotrichiiuo Erysipclotndialcsj’......Ensipciotncha ccac;g . FaccaliUilctuK, Faecqlilalca cyhndrnidcs 0496 018 11 179 61 k__Bactoria;p__Firmictttes.Undassified.UHclassified.Undassif1ed:Undassiiied:Undassi fled 0410 149 0 169 us k_ Bnckna:p_ JJnctenHddes^ _BactenMdia:o_Bmm4d3k's;f_Bactew^ actoroides;s ^Badcroides ,faccis 0.245 507 0 854 87 k__Baeteria:p__Firmicutes.e__Clostridia^__Clostndialcsrf__Rumiiiococcaccacig__Ru minococctKcacjinc1assificd:s__C]osti'idium_kptiim 0.259 28 H.I48 18 k _i Ractoria;p Bnckroidetesx Bactcroidinio Ractcroidalcs;f pdoribactoraccac^ BuhTicinioii3s;s_Bul\Ticimonas j irosa 0 147 346 0 145 84 t_Ba£teria.p__Firmicutes.e__Clustridixu__Chstnduileb.f__Lubactenaceac.g_Fubact ciiaccar_utidassificd:s__LubatfcnareacJiacteriuin_CilKClU(i? 0404 361 0 04

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;k^;wknj.p^nmiicu{rs:c_Ctobtridta,o^ lassi fkd ? ft. 134 51 k__Bacteria.p__Bactcraidetcsic__Bactvioidia.o__Batten 9idaLs.f_R>kcndlaceae.g „.Ali stipcsis AIHxipci ondcrdonkii ' ^: 0 120 1" ft 1 29 '73 to Bactcna;p Bncteroid-ctesx . Bactcroidia.o. Bactcroidalcs.f , Bactcroidaccacig In actcroidcs:? Bactcmidc^ thctaiotaomicmn 0 4JO1 745 0.128 7" to Bacteria:p_ Apyukshspschs ^Actmobacteriato. BfidotacwrialcsJ Bifidobacten acc3c:g_Jlifidnbaek-num:s_BrtidrfwlcnumJnn^ O.lfib 0 1 26 ΞΙ k_Bactcna:p_Bactcn)idi:tes:L‘_Baderoidia;p_Bactcroidaks;f_Rikcnvlljcca..:g__Ah stjpes:s__ASistipes_spJIGB5 1)236 047 0 123 13 k____Bactena;p_Firmicutes:c__(lwndM;o_Clostndi31es;f_L^^ _ Anae Krtigiumi.s_AuaeiOtigmimJacLiti^ ;i Г5 753 (1.122 55 k_B;urlcna:p_Pr0lcobactcn3.c_Ganimaprolei»badenxo_biicrnbue№^ bnctcriaccx'.Liidassiiiicd. Unclassified 0233 501 0 121 66 k_Bacteria.p__Firmiculesx_Clos[ridta.0__Cknridiaks:f_Rimunoe «^^__Ana cromassilibacillus. Unclassified Ί lU f ₅ 0 tn 5 k_J3actcria:p_ PrLMc0bactma:c_Rck^rofcolwteria:0_Bm^ddcriaks:^ cac;g Pnrasiitterelh;?, ParaWtodla yccremcntilw^ 0.169 01 0 in I to Bactcrixp Rncteroidctcsx Bacfcmidia;o Bnctcraidalcs'.f Bacteroidaccactg In uctcroidcsis _ Backmidc?. Anlgaus 0 230 672 ft 106 98 to Rnctcria:p Bnctcroidctcs.c Bacfcroidia.o Baderoidafcsf Rikwlla«ac:g Ali stipcs:s_jMBtipc.vsp^ · O23K 234 ft 105 19 to V,shspd;p Firmiculcs:p Oostridw;o CJostridialcstf , i.aclinospiraccac®. Lach nodubiridinni.s__Clusindiuni^aaediaiolytieuni 0204 04 0.102 22 k_Vdskt1a;r_DZtt1si1skkh_(bk1piga;«.^1oyatShakk;G_kkbt^^ kt Unclassified • 12 - ^r 2b 0.0% 14 k__Baclenu.p__l4rmicutesx_Clostridia.u_Clostridialcs:f_Chstndiaa^ ium:s_ClostndiiiinJcptirm_CAG J7 0.216 42 tl 0% 52 __BactciOidales.f_Odonbactcraccae.g_ BiitMicimoiias.s Butvncimcnms_sp_An62 0.130 67 0 082 06 -¾. Bacrcriatp__Firmicutesx__Ciostndiato__ClustridialesLf_Eubacteriaccae:llnclassific dAJnclassificd 0.250 26 0 064 81 to__Bartering__Bacteroidctesx___Bacteroidia;o__Baetcroidales.f__Rikenelkweauie__Ali slipvs;s„Ali5tipcs_fincgoldii 0235 859 0 046 19 k_Bartcria.p_Bncreroidetes.c_Bacteroidia.o_Bactem^^ __Ali slipes;s Ahstipcs putrcdinis CA(i hl 0.218 202 ft¢1^ 568

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k_Bactena.p_l ^; iraucutos;c__UaL4lko_Luck)ktcdlA^:f_Lactab»il^^ bacillus;» Lactobacillus sain anus ii 228 572 0П5Х 7S1 to Ractonaip _Protcnbacterinx _ GammaprotcobactcruTO Entcrobactcraks.f Entero bnctenaceaag Shigella.Unclassified n?4' 78 0 063 736 Ϊ__Bacwrtaip__FiriMicutes.c_Neg;<h icuiesiUrtdas5iried;UHclassified;Lln^ lassified 0.212 IC 0.063 797 to Bactena:p„Lcntisphacrac;c„,Lcnlisph3ena:o_ . Victimallaksilinclassificd;Undassifi cd. Unclassified ή | a? 645 0.06X 5X4 k__Bacteiia.p__Fifniicutes;c_Clostridia:o__Closttidialcs.f__Lncknospimceac.Lnclassif ied; Unclassified 0 |·.7 l-n? 0 0X0 242 k_Badena.p_Protwbactoria:c_bp5tlunpnH™bactcrb;o_Campylobar^ cohacicraccaa^ lldicobaclcns Helicobacter pnllonim -0.2X8 0.0M1 55u k_Vzhipa.r__Finuicuu-sx__Cbslridia.o__Clostridiaks.f_Rumiuococcaa'ac.g_Sub g1o1shtapts1shyu Subdolip.Kiniiluni variabak 0.313 34? 0.0X2 100 k__Bactcna;p__BaeKroidefcsic__BucKroidia:o_Baetori.Mdak^__Bactomidticeae.g__В acleruidcb.s Bacteroides niassiliensis -UJ 272 0.0X3 306 k__Bactoriaip__Firmicutesic____Clostridiaia __Ckr5tridiala<f_ Rumimrcacca^ cal ibac tcrium; I Indnsss fi al 0 1'' 34 0.0X3 . 360 k__Baacria.p__Ba£totoidetes.c_BacEroidia.o__VshstayakkG_Bactcreidaccae.g__In actvroides:» Bacteroides unifeutms 0.1'X 109 0.0X4 031 k, Vzhgspshr Firm mites x Clostndiazo Clostridialcsif Ruminococcawae.g Ana cmniassihbadihis;" Anauromassilibacillus sp Απ25ϋ 0 244 1 1 2 0 080 645 k___Racteriaip _BacKroida«:c__ Rackroidia:o_ Fhct<:roidaks;f _ Tanncrdlaceae;e_ T annerdlais, Tarmcrdb^sp Ji JJWAA _CTI ; 0.116 28 0 04) 74X k_Baclcrta:p_l4miimes:c_BaCilli;o_Lactobawlkifcs:f_L3ctcte bacillus.” Lx'tobacillus phnsenii 0.2-17 627 0.1 tn 038 k_Bactcrin:p_JBa£teraidctos:c__Bnck^^__Bactcro)dnks.f__Riktiicllaceik\f;__Ali stipes:* Ahsppcs sp CAG 26X ¢. |:r 030 0.1 (is X|4 to VShepa;p I'mnicuresx Clostridia;" Ctostrididcsrf Rutnmococ<Xceac:g Flat onifractor.s Flavom Tractor ^: 0.103 07 0.1()7 791 k_jBa£li-na;p„BadcroidtfL*x_&%to0idia:o__HaciLTO!^ stipes:* Ahsupcs sp SIKSUOZ 0.215 27 0.1 OX 8X7 k_Bactcria;p___Firmicwsx__Clostndiaio___Clostridiak-s;f_Ciostridiaccac.g_BuPrici coccus:»__But) riciciKctis julhcaccorum 0.14 61 o.l [5 59| to__Bactoiia.p__Firniicuies.c__Clostridia.o__ClostrldialcsT__Ruminococc.wac:g__Ge nuHiger.s__Ganniigur_sp_An50 0.16-1 51 0.1 1*! 4(·2 to Vaskhphr Finnicutcsx ClostTidia;o Clostridiaksd' Eubncteriaceaag Eubact erium,s_EubacLenum_rcclalc_CAGJhi (. , ;s 51 0.1311 1X2 to__Baftetia.p_Firmicutos.c__Clostndta.o __Ciostndiak’s.f__Runiinococcacvae.g__Flat onifractoi.s__Flavonirraccor_sp_AiWl oto" 7" 0.130 9X7 k__Vshepa,,p__ΐΊΓηΏαιίνκχ__Clostridia.o__Clostrididies:F_Rmninocoeeaccae:g__Dra naiurtella.Undiissificd 0.109 1.: 0.131 152 to Bactcna;p Protonbacicria; UnelassificdiUndnssificd,(indassiilcd.Unclassified;I Ind unified 0..W 72 0 132 X25

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k Bactana.p_Firnucutcsx Eq5ipdoinchia;o_ Eqsipdotndiak”j.f_.Eqstpdoukha ccaczg MassihomicTobioto.Undassified U.2it3 757 0.135 764 k_Bactena:p _Fi nP1ga1ek:s_ValN Lactabaal laics; f_Lactobaci 11 acuac :g_Lac to bacillus.s Lactobacillus lagmalis 0.120 H5 0.142 632 to Vshspa,p Bactcrpidctcsx Bactcrpidiam BacRroTdalcsif Bactcroubccac.g B ucteroidus.s_Bactcruides_fragilis ; and Π5 ΠΙ (P46 017 to Bactena.p Firmicutcs.c {(lchpf.to CkwlndiaicsJ · (Insindi acene ;g Clostnd mm;s Ctostndium sp A K C 20733 0 ϊ 74 945 0.151 387 to _Badcri.Tp_ Bacteroidetes.c ___Badcroidia:o__Ractcroidalcs;f _ Bactcroidaccnc.g___In actcroidcslhida.ssifkd II (bx 04 0 157 261 k__B.Mvha.p^Bacterwdcks.i^Bactm^__Badeinidaks.r_Riket)dk^ca£'.g_Afi sttpesis Alisn pes sp ^: Aibb 0.155 6?.l 0.1 50 k_ Bnclena;p_ FirmiCHU-sx^ Baciib:n_ i.adobaa]lak<cf_ bchsyubashchPzhsac^^ Lacto bacilluo Lactobacillus reutcri II 24n 012 0 162 187 CJJactena:p_ Baeten4dctcs;c_Biictoroidra;o _m Ja«W£Hdaks:f_^ In 3 ct crcudcs; s_Bad eroidcs Jo rvi 11,074 62 0.164 746 to Bncterixp Firmiculcszc Clo4ridia;o Clostndiaks;!’ Eubact enumzs Eubacteriimi sp Anil I) 175 06 0 16-5 728 k__Baetcna.p__Fimiicutcsx__Clostridial__C[ostridiatesT_EubactaaacMC.g__Eubaci cnunr.s Eubacteriimi sp LpZ 0.188 604 0.167 65| k_VasEspa.r_1atisiK5.s__Baci]ti:o_Lactobacillates.f_Lactobacillaccac:g__Lacto bacillus:s Lactobacillus hekuticui 1P 58 9'0 0.168 52 Bactcna;p. Fimiiciitcsx _Firmicutes qmdassificd.o Firmicntfs tisndassifkdT „ F imicutcs_undnssified:g____Fimuctiks jmdnssilicd.lkdassiFicd II176 5? 0 172 214 to Bnctonnip, Firmicutcs;c Ctastridimo CIostridialcsT Ruminococcaccac-.g, Flav onifractors _ Fla von tfr;actor sp AnIO 0 1167 48 fi 177 671 k___Vaat.tr Ftrmiciites:c__Clostndia.o__Clostridialcszf__Riiminococcaccacai___Psc ndoilasoiiifractor.s Pscudoflavonifractor sp LpKhZ P.I8 547 0.185 364 k_Bacicnu:p_Fimiiciitcs.c__Ci05tridiato_Ckwlndiaics.f__CI»stndiaIcs_ujidxssitiedl. ft Clostndiaks unclassified.s Closiri Jinks bacterium CHKCFHil 0.067 65? 0.1"? 6 37 k_Bactena;p _FtrmiL'ute$.c_('lo«lridia.o__(’loslnd talcs.tJLachnospiraceac.g _fflaut Blauiia sp An8I II 161 327 (J 187 124 to Bactenmp BactciOidctcs;c Bactcroidiaio Bactcroidak'sT Rikendlacenczg L stipes: L'ndassiticd : Π 105 56 (J P2 622 Sactona;p__Fiimicutcs.c__Clostridia.o__Clostridiaks.f__Rnminocaccaccac.g__Psc udoflavonifractor.s Pscudoflaicuiifractor sp An 187 u.234 653 0.206 078 k__Bactcnaip__Bactcrmdvtcs.c__Bactcrendiiizo__Bactcroidales:f_Rikendktccac;g__Ait sttpcs.s Alistipes indisrinctus Ts.273 404 6213 441 k__Bactena;p__Fimnculcs;c__Clirelridiuiu__G^Mndiakn_Lachnospiri№^__Tyzz crdla.s__TyzzcrdlnspAn 114 l· ii 080 4 0218 662 to Vzhkpgpr Firmicntcs.c F3ncilH;o LadobaciHnfcs.f 1 aetdrndllncraeig Lacto hncilluszs Lactobacillus avinrins 0 089 093 0224 3 to Bacteria.p Firmicutcszc .. Badllko _ Lactabadlkaks.f 1 -actobadllaiMezg Lacto badlluszs Lactobacillus inglnwi 0 231 413 0.225 168 k__Bnctcna.p__FitmiLulcs.c__Cbstridia.u__ClosLniitales.f__EubiMcnaceaczg__kubact cnaeeae unclassified.s Eubactcnaceac hacccnum SPKSNIM i to.. Bncteriazp _ Firmicutcszc .. Cfostridiazo , Cbstndiales:f, _ Eubacicrinccnczg _ Ewbact e ri urn. Unclassified k__Battena;|j__H^cniidefcs;c__BMtcmiAa,0_.E^ stipeszs Alistipes sp An54 k, Bactcrinzp Firmicutcsx Closfridiaio Clostndialcs:f Ctostridiaks unclassified: p Closmdiiilcs imdassifiaks Ctostridiaks bacterium GNKSYu06 k_Baeteria:p__Bacteroidetes,c_l^__BackTOidak‘!”,f_C3to _OdonixKler;s__Oduribacler_sp_CAG_788 to Bactcnazp Firmicutcszc, Clostridimo. ClostndtalcsT Ruminococcaccaczg Flav onifhKtors Fbvornfrnctor pfautii 0.105 658 0 0X5 665 1.1.074 65 0 I4R 222 ik 114 29 0 141 62 0.228 612 II 262 $75 0 266 283 0 264 78? 0.270 529 H276 379 k__Bactcria.p__Firmicutes.c__Clustndia.o__Cbstndiaks.f__Closiiidiaccac.g__Closttid imii.s Clostridium sp GAG 678 P.08 1 320 11.278 429 k_ Bacteria:p Bacteroidctcsx Bactermdia.o Bncteroidales.f Rikcndla«ac.g_ AH stipes:s Alistipcs timoncnsis 0 135 766 Π 279 078 k_Backiria.p__Bancroidetes.c_Bactetoidia.o_BacKfOidaleb,f_BacKroidaccac.g_B acteroides.s Baetctoides sp D2 11.1167 534 II248 L' to Radcriazp Firmicutcsx Fq sipJotricluax Eqsipdotridwlesd' Fqstpdotnclia ccwig Masshomicmbirtazs Massiliomicnibiola sp AnXP 0 177 296 0 318 547

Productivity grouping and SIMPER analysis

Taxa were divided into four performance correlation groups: Group 1 taxa were associated with improved FCR and improved BW; Group 2 taxa were associated with improved FCR but reduced BW; Group 3 taxa were associated with increased BW but lower FCR; Group 4 taxa were associated with lower FCR and lower BW. Taxon assignments were made using similarity percentage analysis (SIMPER) (see, for example, Clarke, K. R. (1993). Non-parametric multivariate analyzes of changes in community structure. Australian Journal of Ecology, 18(1), 117-143). Separate analyzes were performed for young animals (day 15 sampling in Example 36) and later phase animals (endpoint sampling in Example 36). Significance was assessed by running 100 SIMPER permutations.

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SIMPER analysis of young birds (starting phase, 15th day of growth) revealed the following taxonomic distributions by productivity correlation groups (Table 26).

Table 26. Taxonomic lineages with productivity correlations

Product Group Taxonomic line Contribution SIMPER Significance k__В acteriajp__В acteroidetesjc__В acteroidiajo__Bacteroi dales; f_Riken ellaceae; g__Alistipes; s__Alist ipesspHGB 0.023 5 Group 1 k__В acteriajp__В acteroidetesjc__В acteroidiajo__Bacteroi dalesjf_TannereIlaceae;g__Parabacteroidesjs__Parabacter oides distasoiiis 0.020 PC0.01 k__Bacteria;p_BactaOidetes;c__Bacteroidiajo__Bacteroi dales;f_Taimerellaceae;g__Parabacteroides;s__Parabacter 0.014 oides merdae k__Bacteriajp__Proteobacteriaceae;c_Gammaproteobacteria;o __EnteiObacterales;f_Eiiterobacteriaceae;g__Escherichia; s__Escherichia_coli 0.018 k__In acteriajp__In acteroidetesjc__In acteroidiajo__Bacteroi Group 2 dales;f_Bamesiellaceaejg__Barnesiellajs_Baniesiella_int estiii iliominis 0.014 k__Bacteria;p_Proteobacteria;c_GammapiOteobacteria;o _EnteiObacterales;f__Enterobacteriaceaejg__Escherichia; s__Escherichia_coli 0.030 P<0.01 k__V acteriajp__V acteroidetesjc__V acteroidiajo__Bacteroi dalesjf__Barnesiellaceaejg__Baniesiellajs__Barnesiellaint estinihommis 0.025 k__V acteria;p__V acteroidetesjc__V acteroidiajo__Bacteroi dales;f_Rikenellaceae;g__Alistipesjs__Alist ipespCHK CI003 0.093 k__In acteriajp__In acteroidetesjc__In acteroidiajo__Bacteroi dales;f_Bacteroidaceae;g__Bacteroidesjs__Bacteroidesx ylanisolvens 0.020 k__Bacteriajp__B acteroidetesjc__Bacteroidiajo__Bacteroi Group 3 dales;f_Rikeaellaceae;g__Alistipesjs__Alist ipesspCHK CI003 k__Bacteiia;p_Bacteroidetes;c__Bacteroidiajo__Bacteroi 0.108 dalesjf__BacteiOidaceaejg__Bacteroidesjs__Bacteroidesjs. ylanisolvens 0.016 к__В acteriajp В acteroidetesjc__В acteroidiajo__Bacteroi dales;f_Bacteroidaceae;g__Bacteroidesjs__Bacteroidesu niformis 0.015 P < 0.05 k__В acteriajp__В acteroidetesjc__В acteroidiajo__Bacteroi dalesjf Barnesiellaceaejg Baniesiellajs Bamesiella sp 0.085 _An22 k__B acteria;p_B acteroidetesjc__B acteroidiajo__Bacteroi dales;f_Bacteroidaceae;g__Bacteroidesjs__Bacteroides_fr 0.075 Group 4 agilis k__B acteriajp__Proteobacteriajc__Epsilonprot eobacteria; o __Campylobacteralesjf__Helicobacter aceaejg__Helicobacter 0.043 erjs__Helicobacterjuillonim k__B acteriajp__PiOteobacteria;c_Gammaproteobacteria;o __Enterobacterales;f_Enterobacteriaceae;g__Shigellajs__ 0.013 Shigella sonnei

SIMPER analysis of birds at the end of their rearing revealed the following taxonomic assignments to productivity correlation groups presented in Table. 27.

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Table 27. Taxonomic assignments of productivity correlation groups

It was observed that different oligosaccharide treatments resulted in distinct shifts in abundance between different productivity correlation groups.

Example 41. Method for modulating taxonomic productivity groups

The birds were fed a diet containing the oligosaccharide preparation from Example 9, where the oligosaccharide identity was selected to increase the abundance of taxa in group 1, group 2 and/or group 3 from example 40.

The birds were fed diets containing the oligosaccharide preparation from Example 9, where the oligosaccharide identity was selected to reduce the abundance of taxa in group 4 from Example 40.

Example 42: Scaling up repeat batches for production

The production scale of oligosaccharide preparations was increased to a 720 L tank reactor with an overhead stirrer. Twelve batch reactions using the scaled procedure derived from Example 9.2 were performed on a 720 L scale. The resulting oligosaccharide preparations were characterized according to predefined quality control acceptance criteria to perform batch qualification and assess process stability.

For twelve batches, process conditions such as temperature, reaction time, and reaction pressure were deliberately varied within a range from the nominal conditions of Example 9.2 to evaluate the sensitivity of the resulting product to reasonable variations in process conditions that might be expected in a typical manufacturing environment. For selected batches, an in situ viscosity probe was used to monitor the viscosity of the reactor contents as a function of time. In some batches, reaction stop times were used for in-process control (IPC) based on continuous viscosity measurement. Quantities of materials, including reactant metering, distillation water, and condensate released, were measured either by mass using load cells on the reactor and auxiliary tanks, or by volumetric flow and time.

The final water content of the reactor product was measured by Karl Fischer titration of a typical aliquot of the reactor contents taken at the end of the reaction, i.e. before pH neutralization and dilution. At a reaction temperature of 120°C, the water content in the reaction product was determined to be 8 and 9 wt.% water, without conversion to dry matter. At a reaction temperature of 130°C, the water content in the reaction product was determined to be from 5 to 7 wt.% water without conversion to dry matter.

The appearance of the resulting oligosaccharide syrup of all batches was determined by visual inspection to be caramel syrup. Total dissolved solids were determined by Karl Fischer titration, residual monomer content, MWn and MWw were determined by HPLC/GPC chromatography, pH was determined by a calibrated pH meter, and anhydro-OP2 content was determined by LC-MS/MS. As shown in table. 29, the following batch characterization data were obtained (N/R = data not reported):

Table 29. Characteristics of oligosaccharide preparations

.rtiya Sol. solid, substance, wt.% pH Residual catalyst DPI, wt.% MWn MWw Anhydro-OP2 content (g anhydro-OP2/g total DP2) 27.1 66.4 N/D N/A 17.5 777 1218 0.84% 27.2 68.8 3.3 N/R 17.9 735 1091 0.91% 27.3 69.4 3.1 0.095 14.8 807 1276 N/R 27.4 71.0 N/D 0.068 15.5 793 1241 1.04% 27.5 70.9 3.2 0.057 15.8 777 1196 1.15% 27.6 70.9 z, z N/A 16.3 773 1170 N/A 27.7 70.7 3.0 n/a 15.7 783 1226 1.13% 27.8 70.5 3.9 n/a 16.1 785 1182 1.09% 27.9 71.1 4.1 n/a 17.1 761 1169 1.09% 27.10 70.4 4.1 n/a 16.3 778 1193 1.15% 27.11 70.5 4.7 n/a 18.6 696 995 1.33% 27.12 70.9 3.9 n/a 16.7 769 1194 1.12%

Example 43. pH Regulation of an Oligosaccharide Preparation

The pH of the oligosaccharide preparation from Example 9.2 at 50 wt.% solids was determined in triplicate by diluting 5.00 ± 0.05 g aliquots of the oligosaccharide preparation in 1.80 ± 0.02 ml of deionized water and vortexing to obtain uniform concentration. The pH value of each aliquot was measured using a calibrated pH meter (VWR, Symphony B30PCI) to obtain an average pH value of 2.4.

To 1.2 kg of the oligosaccharide preparation from Example 9.2 was added 6.53 ml of a 1.0 M aqueous solution of sodium hydroxide. The resulting mixture was stirred vigorously until a homogeneous syrup was obtained with an adjusted pH. The pH of the resulting adjusted syrup was then determined at 50 wt% solids in triplicate as described above to obtain an average pH of 4.1.

The pH adjustment procedure was repeated for replicate batch syntheses at different scales, but with some variations in procedure to provide a basis for the composition of the oligosaccharide product. For one batch, pH adjustment was performed as the final reaction step before diluting the reaction water. In another batch, pH adjustment was performed simultaneously with the dilution step by first dissolving the required amount of base in the dilution water; thus, the base and diluent water were added together to stop the reaction in one step to obtain the final syrup with the desired pH. In another batch, the base came in the form of food grade sodium hydroxide granules. In another batch, 10 ppm food grade silicone emulsion (Dow Xiameter AFE0100) was added to the reaction mixture before dilution and pH adjustment.

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Example 44. Preparation of a glass-like powder composition from an oligosaccharide preparation

Approximately 50 g of the oligosaccharide preparation from Example 9.1 was spread on a drying tray and placed in a forced convection heater at 60° C. to produce a brittle, caramel-colored glass. The glass was removed from the drying tray and ground using a rotary shear mill to obtain a flowable light orange powder. The particle size of the powder was determined by sieving and ranged from 100 to 2000 µm, with 90% of the mass less than 1350 µm. The true density of the coarse crushed powder, determined by a helium pinkometer, was 1.3063 g/ml. The resulting powder was free-flowing.

The forming procedure was repeated using a hammer mill to obtain a fine powder with 90% by weight of the powder having a particle size of less than 196 microns. The true density of the finely ground powder was 1.5263 g/ml. The resulting powder was neither stable nor free-flowing.

DSC measurements were carried out on powders using two temperature cycle programs. In the first program, the temperature was shifted to 160 from 0°C at a rate of 5°C/min, then annealed back to 0°C at a rate of -5°C/min, followed by a final reheat to 160°C. In the second program, the temperature was shifted to 50 from -50°C at a rate of 5°C/min, annealed to -60°C at a rate of -5°C/min, and then heated to 60°C at a rate of 5°C/min. The powder was found to have a glass transition temperature of 20 to 40° C., depending on the residual water content of the solid from 5 to 10 wt.% moisture.

The process of grinding the composition was repeated for each of the oligosaccharide preparations from example 9.2, example 9.3, example 9.4 and example 9.5. The powders were easily redissolved in water and water-alcohol mixtures, but did not dissolve in acetone, methanol, and anhydrous ethanol.

Example 45. Preparation of a powder composition loaded on a carrier

Equal weights of 70 wt.% aqueous syrup of the oligosaccharide preparation from Example 9.2 and diatomaceous earth were combined at room temperature to obtain a stable free-flowing powder. The resulting powder contained about 35 wt.% adsorbed oligosaccharide (based on dry solids) and about 50 wt.% carrier. The particle size distribution of the powder was measured by sieving. 10% by weight of the powder had a particle size of less than 290 microns, 50% by weight of the powder had a particle size of less than 511 microns, and 90% by weight of the powder had a particle size of less than 886 microns. The powder was resistant to segregation and cohesion as determined by standard aeration and compressibility tests. The true density of the resulting powder, measured with a helium pycnometer, was 1.8541 g/ml.

Formation with carrier loading was repeated using feed grade silica to obtain a stable free-flowing powder having an oligosaccharide formulation content of at least 50 wt.% (on a dry solids basis) relative to the final powder. The true density of the resulting powder was 1.5562 g/ml.

Example 46 Preparation of Extruded Solid Form

A solid extruded product was prepared by mixing 20% of the oligosaccharide preparation from Example 9.2 with coarse wheat flour, and preparing the mixture through a twin screw jacketed dry matter extruder to obtain a free-flowing powder with a particle size of 0.2 to 3.0 mm, with 90% by weight less than 2 mm in size. The resulting powder was free-flowing and stable.

Example 47. Preparation of stable powder compositions

Solid compositions, including those of Examples 44-46, were evaluated to determine their stability and hygroscopicity. The powders of Examples 45 and 46 were resistant to segregation and agglomeration, while the powder from Example 44 was unstable to segregation.

Samples of each powder composition tested were placed in sealed environmental chambers at 50% RH and 65% RH for up to two weeks at 25°C. Of the molds tested, some showed little or no weight gain when exposed to humidity, and remained free-flowing after a two-week exposure period. The fine powder of Example 45 was found to be unstable when exposed to moisture.

Example 48. Determination of residual catalyst in oligosaccharide preparations

The residual content of the acid catalyst in the oligosaccharide preparations was determined by ion chromatography. 80 to 100 mg of the powdered oligosaccharide formulation (prepared, for example, as described in Example 44) was dissolved in exactly 1.00 ml and centrifuged to remove particles, if necessary. The resulting solution was analyzed by ion chromatography at 30°C using a Thermo Dionex ICS-3000 system equipped with a conductivity detector, an Ion Pac AS19A 4x250 mm column, an Ion Pac AS19G 50 4x50 mm precolumn and a continuously regenerated CR-ATC anion precolumn using KOH in water as eluent. Elution was performed at 10 mM KOH for the first ten minutes after injection, followed by a gradient elution, increasing linearly to 55 mM KOH after 25 min, then decreasing to 10 mM KOH after 26 min, and maintaining 10 mM KOH until the end of the program.

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For the oligosaccharide preparation of Example 9.2, the concentration of residual catalyst was determined from a standard calibration curve constructed using an authentic sample of (+)camphor-10-sulfonic acid. A typical batch of the oligosaccharide formulation from Example 9.2 was analyzed and the residual catalyst concentration was determined to be 0.62 mg per gram wt.% syrup.

Example 49: Quantitative Analysis of Residual Catalyst Concentration for Batch Acceptance

The catalyst residual determination in Example 48 was compared to the lot acceptance criterion to determine the suitability of the lot for further use. The acceptance limit for the concentration of residual catalyst in the oligosaccharide product formulation was previously established to be <1.0 mg per gram of syrup product. The catalyst residual was measured to be 0.62 mg per gram of syrup product. Thus, the acceptance criterion was met for the tested lot and the lot was accepted for further use.

Example 50 Preparation of product in syrup form

The oligosaccharide preparation of Example 9.7 was adjusted to pH 4.2 with food grade sodium hydroxide according to the procedure of Example 43. The resulting syrup was packaged in a 20-liter bottle with a tamper-resistant cap. Immediately before sealing the container, a 500 g sample was taken and subjected to quality control. The total solids content of the syrup has been verified to be greater than 70% by weight according to FCC Appendix X: Carbohydrates (Starches, Sugars and Related Substances): Total Solids. Reducing sugars have been confirmed to be less than 50% as D-glucose on a dry matter basis according to the FCC Method Appendix X: Carbohydrates (Starches, Sugars and Related Substances): Reducing Sugars Analysis. The sulphated ash content has been verified to be less than 1% of the dry matter weight using FCC Method Appendix II: Physical Tests and Determinations: C. Other: Mineralization Residue (Sulphated Ash), Method II (for liquids). The sulfur dioxide content was less than 40 mg/kg using the optimized Monier-Williams method. The lead content was less than 1 mg/kg using the official international method AOAC 2013.06. The total aerobic microbial count by plate assay was confirmed to be below 1000 CFU/g using the methods described in Chapter 7 of the CMMEF. Total yeast and mold counts were less than 100 CFU/g using the international AACC approved method 42-50. Coliform levels were confirmed to be less than 10 MPN/g using the method described in Chapter 4 of the FDA BAM. E. coli was confirmed to be less than 3 MPN/g using the FDA BAM Chapter 4 method. Salmonella was confirmed to be undetectable in the 25-gram sample according to the FDA BAM Chapter 5 method. Staphylococcus aureus was below 10 CFU/g using the FDA BAM Method, Chapter 12. The color was determined to be caramel by visual analysis. The container was sealed, the remaining sample was frozen and stored for future use, and a certificate of analysis was issued for the resulting batch.

Example 51: Preparation of treated drinking water

Drinking water containing 250 ppm. oligosaccharide drug from example 9.7 was prepared as follows. 37 ml of the oligosaccharide syrup from Example 33 and 40 g of potassium sorbate were gradually added to 50 gallons of drinking tap water in a 55 gallon blue polymer drum. The solution was stirred manually using a paddle mixer for 10 min at room temperature.

The method was repeated without the inclusion of potassium sorbate.

Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are presented by way of example only. The invention is not intended to be limited to the specific examples presented in the specification. Although the invention has been described with reference to the above specification, the descriptions and illustrations of embodiments herein are not intended to be construed in a limiting sense. Numerous variations, changes and substitutions will be apparent to those skilled in the art without departing from the invention. Moreover, it should be understood that all aspects of the invention are not limited to the specific images, configurations or relative proportions set forth herein, which depend on a variety of conditions and variables. It should be understood that various alternatives to the embodiments described herein may be used in the practice of the invention. Accordingly, the invention is also intended to cover any such alternatives, modifications, variations or equivalents.

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Claims (18)

CLAIM 1. A method for increasing the body weight of an animal, including administering to the animal a nutritional composition containing a basic nutritional composition and a synthetic oligosaccharide preparation, where said synthetic oligosaccharide preparation contains at least n fractions of oligosaccharides, each of which has a different degree of polymerization, selected from 1 to n ( fractions DP1 to DPn), wherein n is an integer greater than 3 and wherein each of fractions DP1 and DP2 independently comprises from 0.5 to 15% anhydrosaccharide containing oligosaccharides as measured by mass spectrometry. 2. The method according to claim 1, wherein said body weight of said animal is at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 g greater than said body weight of said animal before administration of said nutritional composition, containing said synthetic oligosaccharide preparation, when measured at least 30, 35, 40, 45, 50, 60, 70, 80 or 90 days after the first administration of said nutritional composition containing said synthetic oligosaccharide preparation, and wherein said animal takes said nutritional composition at least once every 24 hours. 3. A method for reducing the feed conversion ratio of an animal, including administering to an animal a nutritional composition containing a main nutritional composition and a synthetic oligosaccharide preparation, where said synthetic oligosaccharide preparation contains at least n fractions of oligosaccharides, each of which has a different degree of polymerization, selected from 1 to n (fractions DP1 to DPn), where n is an integer greater than 3 and where each of fractions DP1 and DP2 independently comprises from 0.5 to 15% oligosaccharides containing anhydrosubunits, as measured by relative abundance by mass spectrometry. 4. The method according to claim 3, wherein said feed conversion ratio (FCR) of said animal is reduced by at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10% compared to said FCR of said animal prior to administration of said nutritional composition containing said synthetic oligosaccharide preparation, when measured at least 30, 35, 40, 45, 50, 60, 70, 80 or 90 days after the first administration of said nutritional composition containing said synthetic oligosaccharide preparation, and wherein said animal takes said nutritional composition at least once every 24 hours. 5. A method for increasing the efficiency of animal nutrition, including administering to the animal a nutritional composition containing a basic nutritional composition and a synthetic oligosaccharide preparation, where said synthetic oligosaccharide preparation contains at least n fractions of oligosaccharides, each of which has a different degree of polymerization, selected from 1 to n ( fractions DP1 to DPn), where n is an integer greater than 3 and where each of fractions DP1 and DP2 independently comprises from 0.5 to 15% anhydrosaccharide containing oligosaccharides, as measured by mass spectrometry, where nutritional efficiency is the ratio of animal product to the amount of feed consumed. 6. The method according to claim 5, wherein said nutritional efficiency of said animal is increased by at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10% compared to said nutritional efficiency of said animal before using said a nutritional composition containing said synthetic oligosaccharide preparation. 7. The method according to claim 5 or 6, wherein said feeding efficiency of said animal is increased by at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10% compared to said feeding efficiency of said animal to administration of said nutritional composition containing said synthetic oligosaccharide preparation, when measured at least 30, 35, 40, 45, 50, 60, 70, 80 or 90 days after the first administration of said nutritional composition containing said synthetic oligosaccharide preparation, and wherein said animal ingests said nutritional composition at least once every 24 hours. 8. The method according to any of the previous claims, wherein said nutritional composition contains at least 100, 200, 300, 40θ, 500, 600, 700, 800, 900, 1000, 1500 or 2000 ppm of oligosaccharide drug. 9. A method for modulating the growth of at least one type of microorganism in the gastrointestinal tract of an animal, including administering to the animal a nutritional composition containing a basic nutritional composition and a synthetic oligosaccharide preparation, where said synthetic oligosaccharide preparation contains at least n fractions of oligosaccharides, each of which has varying degrees of polymerization selected from 1 to n (fractions DP1 to DPn), where n is an integer greater than 3 and where each of fractions DP1 and DP2 independently comprises from 0.5 to 15% oligosaccharides containing anhydrosubunits, by relative abundance, measured by mass spectrometry. 10. The method according to claim 9, wherein said at least one type of microorganism is an archaea, bacteria, protozoa, virus, bacteriophage, parasite or fungus. - 94 044359 11. The method of claim 10, comprising stimulating the growth of said at least one type of microorganism, and wherein said level of at least one type of microorganism in said gastrointestinal sample is increased relative to the level of said at least one type of microorganism in the gastrointestinal sample. intestinal sample from a comparable control animal that was treated with a comparable nutritional composition not containing the specified synthetic oligosaccharide drug. 12. A method for modulating the expression of at least one microbial protein in the gastrointestinal tract of an animal, comprising administering to the animal a nutritional composition containing a basic nutritional composition and a synthetic oligosaccharide preparation, where said synthetic oligosaccharide preparation contains at least n fractions of oligosaccharides, each of which has a different degree polymerization selected from 1 to n (fractions DP1 to DPn), where n is an integer greater than 3 and where each of fractions DP1 and DP2 independently comprises from 0.5 to 15% oligosaccharides containing anhydrous subunits, by relative abundance, measured by mass - spectrometry. 13. The method of claim 12, wherein said level of said at least one microbial protein in said gastrointestinal sample is reduced compared to said level of said at least one microbial protein in a gastrointestinal sample from said animal prior to administration of said nutritional composition in the specified animal. 14. The use of a nutritional composition containing a basic nutritional composition and a synthetic oligosaccharide preparation, where said synthetic oligosaccharide preparation contains at least n fractions of oligosaccharides, each of which has a different degree of polymerization, selected from 1 to n (fractions from DP1 to DPn), where n is an integer greater than 3 and wherein each of the DP1 and DP2 fractions independently comprises from 0.5 to 15% anhydrosubunit-containing oligosaccharides as measured by mass spectrometry relative abundance to increase the body weight of the animal. 15. The use of a nutritional composition containing a basic nutritional composition and a synthetic oligosaccharide preparation, where said synthetic oligosaccharide preparation contains at least n oligosaccharide fractions, each of which has a different degree of polymerization, selected from 1 to n (fractions DP1 to DPn), where n is an integer greater than 3 and wherein each of the DP1 and DP2 fractions independently comprises from 0.5 to 15% oligosaccharides containing anhydrosubunits, as measured by mass spectrometry relative abundance of the animal's feed conversion ratio reduction. 16. The use of a nutritional composition containing a basic nutritional composition and a synthetic oligosaccharide preparation, where said synthetic oligosaccharide preparation contains at least n fractions of oligosaccharides, each of which has a different degree of polymerization, selected from 1 to n (fractions from DP1 to DPn), where n is an integer greater than 3 and wherein each of the DP1 and DP2 fractions independently comprises from 0.5 to 15% anhydrosubunit-containing oligosaccharides, as measured by mass spectrometry, to improve the nutritional efficiency of the animal. 17. The use of a nutritional composition containing a basic nutritional composition and a synthetic oligosaccharide preparation, where said synthetic oligosaccharide preparation contains at least n fractions of oligosaccharides, each of which has a different degree of polymerization, selected from 1 to n (fractions from DP1 to DPn), where n is an integer greater than 3 and wherein each of fractions DP1 and DP2 independently comprises from 0.5 to 15% oligosaccharides containing anhydrosubunits, as measured by relative abundance by mass spectrometry, to modulate the growth of at least one species of microorganisms in the gastrointestinal animal tract. 18. The use of a nutritional composition containing a basic nutritional composition and a synthetic oligosaccharide preparation, where said synthetic oligosaccharide preparation contains at least n oligosaccharide fractions, each of which has a different degree of polymerization selected from 1 to n (fractions DP1 to DPn), where n is an integer greater than 3 and wherein each of fractions DP1 and DP2 independently comprises from 0.5 to 15% oligosaccharides containing anhydrosubunits, as measured by relative abundance by mass spectrometry, to modulate the expression of at least one microbial protein in the gastrointestinal animal tract. -